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Compiled Comments - Draft ISPMs for country consultation, 2010

DRAFT ANNEX TO ISPM 27:2010Plum pox virus

No. 1. Section 2. Para nber

3. sentence /row/ indent, etc.

4. Type of comment (Substantive,Editorial,Translation)

5. Proposed rewording 6. Explanation 7. Country

1 GENERAL COMMENTS

Substantive 1. It would be useful if the standard were to give some idea re: possible geographic regions where this pest could be established. Information has only been given on where the pest has occurred or currently occurs.

2. Disclaimers for the mention of brand names have not been included in the document. Wherever a brand name is mentioned, a disclaimer must be included as a footnote as agreed in CPM-5.

ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, ST. VINCENT AND THE GRENADINES (SVG), SURINAME, TRINIDAD AND TOBAGO, JAMAICA

2 GENERAL COMMENTS

Substantive Citation of brand names should be revised and modified according to that agree, that is to insert a footnote associated to the brand name cited in the protocol. Paragraph 14 was modified as well as paragraphs where brand names are cited.

ARGENTINA, BRAZIL, CHILE, COSAVE PARAGUAY, URUGUAY

3 GENERAL COMMENTS

Substantive Australia appreciates the work of the Technical Panel to produce this standard. Australia is pleased that its particularly glad that the decision was made to publish

AUSTRALIA

2





those diagnostic protocols that had been approved by the Standards Committee to be released for country comment but had yet to be released on the IPPC website. This should help developing countries.

4 GENERAL COMMENTS

Substantive Insert photos of symptoms Lack of photographs of PPV symptoms is detrimental to the protocol as the description of symptoms is a poor substitute. It would assist diagnosticians if this protocol was a ‘stand alone’ resource.

AUSTRALIA

5 GENERAL COMMENTS

Substantive The document is comprehensive in its recommendations of diagnostic protocols that are suitable for the detection of PPV in general and for the identification of the specific PPV strains of the virus. The document covers both protein/serology-based, and nucleic acid-based diagnostic procedures. Though thorough, there are a number of concerns associated with format and technical details. A major concern is the inconsistency and lack of precise details in describing the various procedures. It is important that the description of procedures be clear and precise in their details to better enable harmonization among relevant parties, to efficiently resolve conflicts arising from contradictory results/data from different parties, and to trouble shoot or constantly improve established procedures.

CANADA

6 GENERAL It is important to specify the PERU, CUBA,

3





COMMENTS microtubes capacity because it is not specified in the document.

Use the appropriate symbol to indicate temperature, since in the document appear two symbols of degree (°°C)

Citations of brand names should be reviewed. That is, insert a footnote associated with the brand referred in the Protocol

Paragraphs where the footnote should be inserted are: 48, 51, 55, 81, 84

There is information in some parts of the text without bibliographic citation (e.g., paragraph 2, losses caused by the virus)

PPV aphid vectors should be included in paragraph 4.

In paragraphs 35 and 70 recommended doses for RNA should be added.

In paragraph 50 the primers for internal control should be included to have higher level of confidence

PANAMA

7 GENERAL COMMENTS

Es importante especificar la capacidad de los microtubos ya que no está especificada en el documento.

COSTA RICA

4





Utilizar el símbolo adecuado para indicar la temperatura, ya que en el documento se encuentran dos símbolos de grado (°).

Deberían revisarse las citas de las marcas comerciales de acuerdo a lo definido. Esto es, insertar una nota al pie asociada a la marca citada en el protocolo. Los párrafos donde debería insertarse el pie de página serían: 48, 51, 55, 81, 84.

Hay información citada en algunas partes del texto que no tiene la cita correspondiente (e.g., párrafo 2, pérdidas causadas por el virus)

Se deberían incluir en el párrafo 4, las especies de áfidos vectores del virus.

En los párrafos 35 y 70 se deberían agregar las dosis de RNA recomendada.|

En el párrafo 50 se deberían incluir los primers para el control interno a fin de dar mayor nivel de confianza.

8 GENERAL COMMENTS

Mexico agrees and supports approval of a new annex to ISPM 27:2010 Plum pox virus, as a new diagnostic protocol for regulated

MEXICO

5





pests.

Use the appropriate symbol to indicate temperature, since in the document appear two symbols of degree (°°C)

There is information in some parts of the text without bibliographic citation (e.g., paragraph 2, losses caused by the virus)

PPV aphid vectors should be included in paragraph 4.

In paragraphs 35 and 70 recommended doses for RNA should be added.

In paragraph 50 the primers for internal control should be included to have higher level of confidence

9 GENERAL COMMENTS

Pag. 1 in: Consultation on technical level.

Pag.2 same section

Editorial Drs. M. Cambra....(IVIA, Ctra. Moncada-Náquera, km 4.5, ......del CNR, Bari, Italy), Dr. T. Candresse...

Györgyi, Hungary), Dr. B. Rodoni (Dept.of Primary…and Dr. C. Varveri (Benaki Phytopathological Institute, Kifissia…

Please, modifyDrs. km 5 Dr.

Dept.

Institute

EU, EPPO

10 GENERAL COMMENTS

Dr. L. Palkovics (Agricultural

Editorial Dr. L. Palkovics (Agricultural Biotechnology Centre, Gödöllő, Hungary)

The name of the town was not correct

EU, EPPO

6





Biotechnology Centre, Györgyi, Hungary)

11 GENERAL COMMENTS

Substantive To add the LAMP method in molecular test known as the simple and sensitive diagnostic tool.

Reference;

Varga, D. James. 2006. Use of reverse transcription loop-mediated isothermal amplification for the detection of Plum Pox Virus. Journal of Virological Methods,138:184-190.

JAPAN

12 GENERAL COMMENTS

Change MgC12 to MgC12 of the whole document

Appropriate formatting of subscript

SEYCHELLES

13 GENERAL COMMENTS

Paragraph 26

The DAS-ELISA method is less accurate in comparison to DASI-ELISA

The relevance of considering a less accurate method, when more accurate methods such as DASI-ELISA are available should be clarified

SOUTH AFRICA

14 GENERAL COMMENTS

When discussing diagnostic methods, it would be better to state the advantages and disadvantages of each method. This would be helpful for countries to choose the most appropriate method available to them. We suggest a comparison table containing tests, reliability, cost, ease of use information.

Since this protocol was written, the antibody listed under serological detection has not been used as often. The DP should list other antibodies as well so it does not seem like the IPPC is endorsing one antibody over the other.

USA

7





15 1. Pest Information [2] Line 6 Substantive Add: Reference to the figure For verification CHINA

16 1. Pest Information [2] 2nd sentence

Editorial The disease, caused by Plum pox virus (PPV), affects plants of the genus Prunus;

Clearer text EU, EPPO

17 1. Pest Information [2] Sentence 1

Editorial Sharka (plum pox) is one of the most economically important serious diseases of stone fruit in terms of agronomic impact and economic importance.

Clearer wording JAMAICA


Editorial …. affects the genus Prunus; it , and is particularly…..

Clearer wording JAMAICA

19 1. Pest Information [2] Line 6 Substantive Add: Reference to the figure For verification KOREA


Sentence 3

Sentence 3

Editorial

Editorial

Substantive

The Sharka (plum pox) is one of the most serious diseases of stone fruit in terms of and it has great agronomic and economic impact.

It is e stimated that costs associated with of sharka management worldwide since the 1970s exceed 10 000 million euros

(reference)

To improve the redaction.

To improve the redaction.

It is necessary put the reference at end of this paragraph to support it.

MEXICO

21 1. Pest Information [2] para Substantive Sharka (plum pox) is one of the most serious diseases of stone fruit in terms of agronomic impact and economic importance. The disease, caused by Plum pox virus (PPV), affects the genus Prunus.; it is particularly detrimental in apricot (P. armeniaca), European plum (P. domestica), Japanese plum (P.salicina), and peach (P. persicae) because it reduces quality and causes premature fruit drop. Estimated costs associated with sharka management

Background information needs to be simplified. The document does not discuss all posible hosts, so partial information on some should not be included.

Estimated costs information is not

USA

8





worldwide since the 1970s exceed 10,000 million euros

needed in a diagnostic protocol.


Editorial Sharka was first reported in eastern Europe, in Bulgaria in 1917–1918, and was described as a viral disease in 1932.

Naming the country should be enough

AUSTRALIA


Substantial Sharka was first reported on plums in eastern Europe, in Bulgaria, in 1917–1918, and was later described….

Describes how the virus (Plum pox virus) got its name.

JAMAICA


1. Editorial2. Editorial3. Technical4. Technical5. Technical

PPV is a member of the genus Potyvirus in the family Potyviridae. The virus particles are flexuous rods of about 700 nm × 11 nm and are composed of a single-stranded RNA molecule consisting of almost 10 000 nucleotides coated by up to 2 000 subunits of a single coat protein (García and Cambra, 2007). PPV is transmitted in the field by aphids in a non-persistent manner, but movement of infected propagative plant material has been and is the main way in which sharka PPV is spread over long distances.

1. “of” added for clarity2. “and are” added to be more specific3. information is required re: the specific aphids that are vectors of PPV4. More clarity is needed. Does this mean that the virus is non-persistent in the aphid or non-persistent in the host plant?5. to be more technically correct the name of the virus and not the name of the disease should be used./ Need to be more technically correct. It is the virus that is transmitted and not the symptoms of the disease, which is usually what the common name refers to.

ANTIGUA AND BARBUDA, BARBADOS, CRWS, DOMINICA, SVG, SURINAME


Substantive. Clarification required

PPV is transmitted in the field by aphids in a non-persistent manner, but movement of infected propagative plant material has been and is the main way in which sharka is spread over long distances.

Information is required re: the specific aphids that are vectors of PPV.

TRINIDAD AND TOBAGO


Substantive. Clarification required

PPV is transmitted in the field by aphids in a non-persistent manner, but movement of infected propagative plant

More clarity is needed. Does this mean that the virus is non-persistent in the aphid or non-

TRINIDAD AND TOBAGO

9





material has been….. persistent in the host plant?


Substantive PPV is transmitted in the field by aphids in a non-persistent manner, but movement of infected propagative plant material has been and is the main way in which sharka PPV is spread over long distances.

Need to be more technically correct. It is the virus that is transmitted and not the symptoms of the disease, which is usually what the common name refers to.

TRINIDAD AND TOBAGO, JAMAICA


Editorial The virus particles are PPV are flexuous rods about approximately 700 nm × 11 nm, and are composed of a single-stranded RNA molecule consisting of almost 10 000 nucleotides. The RNA is encapsidated by a single coat protein consisting of coated by up to 2 000 protein subunits of a single coat protein (García and Cambra, 2007).

Clearer wording, scientific writing.

JAMAICA


editorial Sharka was first reported in eastern Europe in Bulgary on 1917-1918, and it was described as a viral disease in 1932.

To improve the redaction MEXICO

30 1. Pest Information [4] Last sentence

Substantive Delete This information is not needed by experts in the field. Protocols are not the place for a lot of background information.

USA

31 1. Pest Information [5] Editorial PPV isolates can be classified into seven types or strains: D (Dideron), M (Marcus), C (Cherry), EA (El Amar), W (Winona), and Rec (Recombinant) and T (Turkish) (Candresse and Cambra, 2006; James and Glasa, 2006; Ulubaş Serçe et al., 2009). Most...

Correct punctuation ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME, TRINIDAD AND TOBAGO

10





32 1. Pest Information [5] Line 2 Editorial (El Amar), W (Winona) and, Rec (Recombinant) and T (Turkish) (Candresse and Cambra, 2006; James

Technically correct English.

The use of “and” is inappropriate because there is continuous listing of the PPV isolates and thus insertion of a coma necessary

EU, EPPO

SEYCHELLES, SOUTH AFRICA

33 1. Pest Information [5] 5th sentence

Editorial El Amar isolates (PPV EA) EA isolates are geographically restricted to Egypt

Consistent with the way other strains are referred to.

EU, EPPO


Editorial Cherries were not considered a host of PPV for a long time. However, but a number of PPV isolates infecting sour cherry (P. cerasus) and sweet cherry (P. avium) have since been identified in several European countries and Turkey.

Joining of two sentences with same subject.

JAMAICA


Substantive However, a number of PPV isolates infecting sour cherry (P. cerasus) and sweet cherry (P. avium) have been identified in several European countries and Turkey.

Since now, the presence of PPV from sour and sweet cherries are not reported in Turkey. It is therefore, the name of Turkey should be taken out from the statement.

TURKEY

36 1. Pest Information [5] Para Substantive - We suggest to identify strains in order of aggressiveness. This is important information for NPPOs for epidemiology and eradication purposes. For example, in the US, PPV had 500 m buffer, If it was PPV-M (more aggressive), it might have been a 1,000 m buffer. This is also important information for trading partners.

USA

37 1. Pest Information [6] Sentence Substantive Further information about PPV, Add PaDIL Plant Biosecurity AUSTRALIA

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1 including illustrations of disease symptoms, can be found in CABI (2008), EPPO (2004), EPPO (2006), PaDIL (PBT), and García and Cambra (2007).

ToolBox website for a set of photos and further information on PPV http://www.padil.gov.au/pbt/index.php is general reference, http://www.padil.gov.au/pbt/index.php?q=node/46&pbtID=136 is specific plum pox reference

38 1. Pest Information [6] Para Substantive Further information about PPV, including illustrations of disease symptoms, can be found in CABI (2008), EPPO (2004), EPPO (2006) and Garcia and Cambra (2007), and PPQ photo gallery website.

This website contains a nice collection of symptoms pictures, gathered from worldwide experts.

USA


Substantive Add pictorial of disease symptoms Will be more appropriate and add value to the text

SEYCHELLES


Substantive Add illustration of disease symptoms Although reference to CABI is given here, some illustrations of the disease symptoms would be more appropriate and add value to the descriptions.

SOUTH AFRICA

41 2.Taxonomic Information

[7] Synonyms: Sharka virus

Editorial General synonym: Sharka virus Plural is not necessary if only the general synonym is mentioned

EU, EPPO

42 3.Detection and Identification

[8] title Substantive Delete: “and Identification” NO reference to identification, only positive or negative

CHINA


[8] 1st sentence

Editorial Under natural conditions, PPV readily infects fruit trees of the genus Prunus used as commercial varieties or

Clearer wording

EPPO

http://www.padil.gov.au/pbt/index.php?q=node/46&pbtID=136

http://www.padil.gov.au/pbt/index.php?q=node/46&pbtID=136

http://www.padil.gov.au/pbt/index.php

http://www.padil.gov.au/pbt/index.php

12





3rd sentence

Editorial

rootstocks: apricot, Chinese wild peach (P. davidiana), European and Japanese plums, peach, Chinese wild peach (P. davidiana), Mahaleb cherry (P. mahaleb), Marianna plum (P. marianna) and Myrobalan flowering plum (P. cerasifera).

The virus also infects many wild or and ornamental Prunus species such as western sand cherry (P. besseyi), purple-leaved sand cherry (P. cistena), dwarf flowering almond (P. glandulosa), Damson plum (P. insititia), cherry laurel (P. laurocerasus), blackthorn (P. spinosa), nanking cherry (P. tomentosa) and flowering almond (P. triloba).

Clearer wording


[9] para substantive Insert images Images would assist in interpretation of the text

AUSTRALIA


[9] Sentence 1 and 2

Editorial Under natural conditions, PPV readily infects fruit trees of the genus Prunus used as commercial varieties or rootstocks: apricot ( P. armeniaca ) , European and Japanese plums ( P. domestica and salicina ) , peach, Chinese wild peach (P. davidiana), Mahaleb cherry (P. mahaleb), Marianna plum (P. marianna) and Myrobalan flowering plum (P. cerasifera). Sour and sweet cherries ( P. cerasus and avium ) , and almond (P. dulcis) may be infected occasionally.

Include binomial names of all host species even though some have appeared before (for consistency)

AUSTRALIA


[9] 1st sentence

Technical Add more precisions at the end of the sentence [after "…(P. cerasifera)"]:

To complete the list

EU

13





3rd sentence

Editorial

.. and interspecific hybrids between these species often maintained in botanical repositories or used as root-stock.

The virus also infects many wild or and ornamental Prunus species such as western sand cherry (P. besseyi), ..

Clearer wording


[9] Sentence 1

Substantive Add more precisions alter (P. cerasifera):and interspecific hybrids between these species often maintained in botanical repositories and/or used as root-stock.

To complete the list EPPO


[9] Last sentence

Technical Nicotiana benthamiana, N. glutinosa, N. clevelandii and , Pisum sativum and Arabidopsis thaliana .

Currently it is possible to infect with PPV this experimental herbaceous host that is being used for basic researching on resistance against PPV.

EU, EPPO


[9] 1st Sentence

Substantive …of the genus Prunus used as commercial varieties or rootstocks: apricot, European and Japanese plums, peach, Japanese apricot ( P. mume ) , Chinese wild peach (P. davidiana) ,...

Under natural condition, PPV was detected from Japanese apricot trees in Japan in 2009 (Maejima et al., Journal of General Plant Pathology, 2010, 76: 229-231).

JAPAN


[9] Substantive Add: Japanese Apricot ( Prunus mume ) Detected in 2009. KOREA


[10] 1. Editorial2. Editorial3. Editorial4. Technical

…. The alcohol or spirits produced from this these fruits are unmarketable owing to an undesirable flavour. In severe cases the diseased fruits drop prematurely from the tree. In general, the fruits of early bearing cultivars show more marked symptoms than those of late cultivars.

1., 2. And 3. For proper sentence construction;

4. for clarity

ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME, JAMAICA

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[10] Sentence 9 Editorial …. The alcohol or spirits produced from this these fruits are unmarketable owing to an undesirable flavour

For proper sentence construction. TRINIDAD AND TOBAGO


[10] Sentence 11

Editorial In general, the fruits of early cultivars show more marked symptoms than those of late cultivars.

For proper sentence construction. TRINIDAD AND TOBAGO


[10] Sentence 11

Editorial In general the fruit of early bearing cultivars show more marked symptoms than those of late cultivars.

For clarity. TRINIDAD AND TOBAGO


[10] Sentence 1

Substantive Complete the sentence:Leaves, bark, petals, fruits, stones and shoots

To complete the list. The symptoms on the bark of GF 31 have diagnostical value.

EU, EPPO


[10] 2nd sentence +

Insert new sentence

Insert new sentence

Editorial

Technical

Technical

… They are particularly clear on leaves in springtime and include mild light-green discoloration; chlorotic spots, bands or rings; vein clearing or yellowing; or even leaf deformation. … under the discoloured rings. Some fruit deformations, especially in apricot and European plum, are similar to those caused by Apple chlorotic leaf spot virus also named “pseudo plum pox”. Stones from diseased apricot fruits show typical pale rings or spots. Diseased fruits may show internal browning and gummosis of the flesh and reduced quality. P. cerasifera cv. GF 31 indicator shows rusty brown corking and cracking of the bark. The alcohol or spirits produced from this fruits are unmarketable owing to an undesirable flavour. In severe cases the diseased fruits drop prematurely from

Unnecessary words

Pseudo plum pox can cause similar symptoms on apricot and European plum fruits.

These are diagnostically important symptoms.

Clearer wording (reordered text)

EU

15





Editorial the tree. In general the fruit of early cultivars show more marked symptoms than those of late cultivars. Stones from diseased apricot fruits show typical pale rings or spots. The alcohol or spirits produced from this diseased fruits are unmarketable owing to an undesirable flavour.


[10] Last sentence

Substantive Before the last sentence add “It should be noted that symptoms development and intensity strongly depends on host plant and climatic conditions. In particular in cold climates, latency may be prolonged for several years.”

The original para may leave the false impression that symptoms appear early and are easily detectable.

EU, EPPO


[10] 2nd sentence

Editorial PPV symptoms may appear on leaves, petals, fruits and stones. They are particularly clear on leaves in springtime and include mild light-green discoloration; chlorotic spots, bands or rings; vein clearing or yellowing; or even leaf deformation. Some of these leaf symptoms are similar to those caused by other viruses, such as American plum line pattern virus. Flower symptoms can occur on petals (discoloration) of some peach cultivars when infected with PPV-M or in P. glandulosa infected with PPV-D. Infected fruits show chlorotic spots or lightly pigmented yellow rings or line patterns. Fruits may become deformed or irregular in shape and develop brown or necrotic areas under the discoloured rings. Stones from diseased apricot fruits show typical pale rings or spots.

Unnecessary words

Clearer wording (reordered text)

EPPO

16





Diseased fruits may show internal browning and gummosis of the flesh and reduced quality. The alcohol or spirits produced from this fruits are unmarketable owing to an undesirable flavour. In severe cases the diseased fruits drop prematurely from the tree. In general the fruit of early cultivars show more marked symptoms than those of late cultivars. Stones from diseased apricot fruits show typical pale rings or spots. The alcohol or spirits produced from this diseased fruits are unmarketable owing to an undesirable flavour.


[10] Insert new sentence

Substantive under the discoloured rings. Some fruit deformations, especially in apricot and European plum, are similar to those caused by Apple chlorotic leaf spot virus also named “pseudo plum pox”. Stones from diseased…

Pseudo plum pox can cause similar symptoms on apricot and European plum fruits.

EPPO


[10] Insert new sentence

Substantive Stones from diseased apricot fruits show typical pale rings or spots. P. cerasifera cv. GF 31 indicator shows rusty brown corking and cracking of the bark. Diseased fruits may

These are diagnostically important symptoms.

EPPO


[10] Sentence 2

Editorial ….. chlorotic spots, bands or rings; vein clearing or yellowing; or even leaf deformation.

“Even” is not necessary JAMAICA


[10] Sentence 9

Editorial The alcohol or spirits produced from this these fruits are ….

‘this’ is a singular term JAMAICA


[10] Substantive To add illustrations of disease symptoms and similar diseases in this section or rearranged to attachments.

Disease symptoms cannot be completely expressed by words only.

JAPAN

17






[10] Substantive Add some pictures/references of the symptoms

Needs to give

Reader/user friendly SEYCHELLES


[10] Substantive Also add illustrations of disease symptoms

Illustrations of symptoms would add more value to the descriptions.

Pseudo-pox can be an example where symptoms can be misleading

SOUTH AFRICA


[10] Sentence 8

Sentence 9

Editorial Infected Diseased fruits may show internal browning and gummosis of the flash and reduced quality.

The alcohol or spirits produced from these this fruits are unmarketable owing to an undesirable flavour.

To improve the redaction


MEXICO


[10] First sentence

Second sentence

Substantive

Substantive

PPV can be visually diagnosed using symptoms that may appear on leaves, petals, fruits and stones in the field (see reference list at the end of the Pest Information section).

They are particularly usually clear on leaves in springtime early growing season and include mild light-green discoloration; chlorotic spots, bands or rings; vein clearing or yellowing; or even leaf deformation.

PPV does not necessarily show symptoms on all of the plant parts listed. The word “field” is more appropriate and accurate for what the sentence is trying to convey.

Sometimes, symptoms can not be seen at all. The term “springtime” is relative depending on where you are, based on latitude (in Prunus growing region).

USA

USA

68 3.Detection and [11] Substantive Appropriate sample selection is critical To specify that it is not ARGENTINA,

18





Identification for PPV detection. If typical symptoms are present, collect flowers, leaves or fruits showing symptoms. In symptomless plants samples should be taken from at least one-year-old shoots with mature leaves or fully expanded leaves (because detection in less than one year old shoots is not reliable) collected from the middle of each of the main branches.

recommended taking samples from less than one year old shoots.

BRAZIL, CHILE, COSAVE PARAGUAY, URUGUAY


[11] New 2nd sentence

Substantive Appropriate sample selection is critical for PPV detection. Sampling should take into account virus biology, and the local climatic conditions including the weather conditions of that year. If typical symptoms are present, collect flowers, leaves or fruits showing symptoms.

New text from CPM Panel. Technically improved sampling guidance.

EU, EPPO


[11] 4th sentence

Editorial

Technical

the main branches. Sampling must should not be done avoiding months with the highest temperatures (temperatures above x o C should be avoided).

Advice on when and what to sample

Question to steward or M. Cambra: can the critical maximum temperature be stipulated? Important for providing further guidance.

EPPO


[11] 3rd last row

Substantive not more than 710 days before processing

More feasible e.g. if samples need to be moved to another lab for further testing (e.g. with PCR); from practice it is clear that after 10 days tests can be successfully performed.

Note to global steward: The final

EPPO

19





sentence could be: “7-10 days” (see line 21 answer on this issue)


[11] 1. Editorial2. Editorial3. Technical

Appropriate sample selection is critical for PPV detection. If typical symptoms are present, collect flowers, leaves or fruits showing symptoms. In symptomless plants, samples should be taken from at least one-year-old shoots with mature leaves or fully expanded leaves collected from the middle of each of the main branches. Sampling must be done avoiding should not be done during months with the highest temperatures.

1. proper sentence structure2. sentence cumbersome3. This is ambiguous. Included in this paragraph should be a specific temperature range.

ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME


[11] Sentence 4 Editorial Sampling must be done avoiding should not be done during months with the highest temperatures.

Proper sentence structure. TRINIDAD AND TOBAGO


[11] Sentence 4 Substantive. More specific information needed

Sampling must be done avoiding months with the highest temperatures.

This is ambiguous. Included in this statement should be a specific temperature range.

TRINIDAD AND TOBAGO, JAMAICA


[11] Sentence 4

Substantive Sampling must be done avoiding months with the highest temperatures. The preferred period for sampling is in the spring, early summer, and fall months as higher titres of the virus tend to occur in the cooler months of the growing season, producing more reliable results.

The statement “Sampling must be done avoiding months with the highest temperature.” is imprecise and unclear and should be deleted and replaced by a new sentence.

CANADA


[11] 4th sentence

Editorial

Technical

… the main branches. Sampling must should be done avoiding months with the highest temperatures above ….. (to be specified by steward).

Advice on when and what to sample Question to steward: can the critical maximum temperature be stipulated? Important for providing further guidance.

EU

20





Taking into account the variation between regions it is better to indicate the range rather than stating high temperature that can be subjective.


[11] After 4th row

Substantive Add ranges of temperatures Comment to steward: Taking into account the variation between regions it is better to indicate the range rather than stating high temperature that can be subjective.

EPPO


[11] Sentence 4

Editorial Sampling must avoid be done avoiding months with the highest high temperatures.

Simplifying the text AUSTRALIA


[11] Sentence 4

substantive Test results may be invalid if samples are taken in months when the average temperature exceeds xxx degrees Celsius/Centigrade. Sampling must be done avoiding months with the highest temperatures.

To remove subjectivity about ‘high temperatures’

AUSTRALIA


[11] Sentence 5

Editorial Plant material should preferably be collected from the internal parts of the canopy of the tree canopy.

Clearer wording AUSTRALIA


[11] Last sentence

substantive In winter dormant buds or bark tissues from the basal part of twigs, shoots or branches, or complete spurs or dards can be selected

What are ‘dards’? – not in any dictionary. Use a more common term or delete

AUSTRALIA


[11] Last sentence

Technical or complete spurs or dards can be selected.

Unknown term, spur is technically correct.

EU, EPPO


[11] 3rd last row

Substantive not more than 7 to 10 days before processing

More feasible e.g. if samples need to be moved to another lab for further testing (e.g. with PCR); from practice it is clear that after 10 days tests can be successfully performed.

EU

21






[11] Sentence 3

Sentence 7

Last sentence

Editorial

Editorial

Editorial

In symptomless plants, samples should

In summer and autumn, mature leaves

In winter, dormant buds or bark tissues from the basal part of twigs, shoots or

Addition of commas to assist clarity of text.

EU, EPPO


[11] New paragraph

Substantive 11 a. In some circumstances (e.g. during the routine diagnosis of a pest widely established in a country) multiple plants may be tested simultaneously using a bulked sample derived from a number of plants. The decision to test individual or multiple plants would depend on the virus concentration in the plants and the level of confidence required by the NPPO.

Discusses the problem of sampling.

To allow for testing more than one plant at once.

NEW ZEALAND, SOLOMON ISLANDS


[11] Sentence 3

editorial ....with the highest temperatures (refer to ISPM 31 for sampling methods).

A reference to ISPM 31 on sampling methods would be appropriate since the sampling procedures themselves are not specified in the paragraph.

SOUTH AFRICA


[11] Sentence 3

Substantive Sampling must be done avoiding months with the highest temperatures ISPM 31:2006.

Need reference regards to temperature regime and symptoms expression

More clearer guidance in terms of temperature in relation to sample

A reference to ISPM 31on sampling methods would be appropriate since the sampling procedures themselves are not specified in the paragraph

Clarity

Clarity

SEYCHELLES

22





collection is require especially for non-temperate areas.


[11] 7th sentence

Add new 8th sentence

Substantive

substantive

In summer and autumn mature leaves and the skin of mature fruits collected from the field or packinghouses can be used for analysis.

Testing of leaves should be avoided in autumn unless it is absolutely necessary because of the chance for false positives in ELISA.

Experts should not look at autumn leaves because as temperatures decline, changes in leaves cause all types of problems in ELISA tests, which is what a lot of countries use. This paragraph makes it sound like it is aceptable to test in autumn when it should only be done as a last ditch resort.

Same as above. It may be hard to find a lot of fruit in autumn, unless it is in packing houses. If testing from packing houses, it would imply that fruit is still being collected from the fiels at this time.

USA

USA


[12] Editorial

Substantive

Detection and identification of all PPV isolates can be achieved using biological, serological or molecular tests following the flow diagram shown in Figure 1. These tests are the minimum requirements to detect and identify PPV (e.g. during routine diagnosis of a pest widely established in a country), but further tests may be required where the national plant protection organization (NPPO) requires additional confidence or to identify the strain of PPV. In all cases, positive and negative controls must be included in the tests. The recommended techniques are described in the following sections.In cases where the disease is not known

The flow diagram shown in Figure 1 is too general, confusing and does not reflect all possible options described in the text

In these cases this is special important to use more sensitive techniques to confirm the


23





Substantive

to occur or for imported material, confirmations should be made with more sensitive technique.

diagnostic.

The flow diagram shown in Figure 1 is too general, confusing and does not reflect all possible options described in the text.


[12] Detection and identification of all PPV isolates can be achieved using biological, serological or molecular tests following the flow diagram shown in Figure 1. These tests are the minimum ...

Se propone eliminar el diagrama de la figura 1 por no reflejar las opciones que se mencionan en el texto.

COSTA RICA


[12] Detection and identification of all PPV isolates can be achieved using biological, serological or molecular tests following the flow diagram shown in Figure 1. These tests are the minimum ...

It is proposed to delete the diagram of Figure 1 because does not reflect the options mentioned in the text.

LARWS, PERU


[12] Line 2

Line 5

Editorial Change “minimum requirement” to “ recommended methods”

Change: “additional confidence” to “further verification”

More appropriate in expresssion CHINA, THAILAND


[12] All Substantive Comment to global steward: Can you ever be confident enough to base a positive identification on ‘typical symptoms’ alone? Not sure if this has already been

EPPO

Biological indexingGraft inoculation of woody

indicator plants (P. persicae cv. GF305, P. persicae P.

davidiana cv. Nemaguard, or P. tomentosa)

Serological testDASI-ELISA with 5B-IVIA universal monoclonal antibody, or DAS-ELISA with 5B-IVIA or polyclonal antibodies; or

Molecular testRT-PCR or IC-RT-PCR (P1/P2 or 3′NCR primers), Co-PCR (P10/P20/P1/P2 primers) and hybridization with

universal probe, or real-time RT-PCR

No symptoms

Plum pox virus not present

Plum pox virus present

Typical symptoms

Plum pox virus present

Positive Negative

24





discussed but it may be worth considering again.The best would be to confirm “typical symptoms” by serological or molecular tests such as is suggested in the EPPO/OEPP (2004) protocol.


[12] Line 2

Line 5

Substantive Change “minimum requirement” to “recommended methods”

Add: “further verification” Delete: “additional confidence”

indexing may not be used in every countries anymore due to development of sensitive detection methods such as PCR etc.

KOREA


[12] 2nd sentence

Substantive These tests are the minimum requirements recommended method to detect and identify PPV…

All tests are not always required to detect.

JAPAN


[12] All Substantive - Comment to TPDP: How reliable are typical symptoms as a basis for positive identification. Are they reliable enough?

EU


[12] Figure 1 Substantive To start with the plant parts involved before proceeding with the respective testing ie for budwood, biological indexing is required. But for fruit or flower samples, serological and molecular tests are recommended testing pathway and not biological indexing.

The title of Figure 1 reflected as mínimum requirements for the detection and identification of PPV. However, biological indexing is only applicable for budwood and not for other plant parts. Hence, the inclusión of simple types before the testing pathway would be a clearer flow diagram of the mínimum testing requirements.

SINGAPORE


[12] Sentence 2

Editorial ....required where the nNational pPlant pProtection oOrganization (NPPO) requires...

Correct formatting and consistency


25





99 Figure 1 [13] Substantive Figure 1: Minimum requirements for the detection and identification of Plum pox virus (e.g. during routine diagnosis of a pest widely established in a country).

See comment on para 12

Ver párrafo 12See paragraph 12

ARGENTINA, BRAZIL, CHILE, URUGUAY, COSAVE PARAGUAYCOSTA RICA, PERU, PANAMA, CUBA

100 Figure 1 [13] 1. Second row, first box

2. After second row

Substantive

Substantive

1. Typical/Atypical symptoms.

2. New third row, new box: Confirmed by Serological test or Molecular test

1. Basing the movement of germplasm across borders simply on biological indexing seems inadequate. Add the word “atypical” beside typical in the first box as atypical symptoms on biological indicators are not addressed in the flow diagram. Depending on the environmental conditions atypical symptoms may be observed.

2. Modify the flow diagram by adding a new box to confirm biological symptoms with either the universal PPV ELISA or Universal PPV RT-PCR. It might be prudent to recommend confirmation of both typical and atypical symptoms on biological indicators, by EM, serology-based, or nucleic acid-based techniques if available.

CANADA

101 Figure 1 [13] Line 1 Substantive Change Title of Figure “minimum requirement” to “ recommended methods ”

More appropriate in expresssion

indexing may not be used in every

CHINA, THAILAND

KOREA

26





countries anymore due to development of sensitive detection methods such as PCR etc.

102 Figure 1 [13] - Substantive Biological indexing Graft inoculation of woody indicator plants (P. persicae cv. GF305, P. cerasifera cv. GF31 , P. persicae P. davidiana cv. Nemaguard, or P. tomentosa)

The use of P. cerasifera cv. GF31 is not very common nevertheless it is possible to use. Could be included.

EU, EPPO

103 Figure 1 [13] Last sentence

Editorial ...established in a country). Full stop missing EU, EPPO

104 Figure 1 [13] Box 2 Editorial Serological testDASI-ELISA with 5B-IVIA universal monoclonal antibody, or DAS-ELISA with 5B-IVIA or PPV specific polyclonal antibodies; or …..

Needs to be clear that the polyclonal antibodies are specific in detecting all PPV

JAMAICA

105 Figure 1 [13] Substantive

Substantive

To add sap inoculation for herbaceous plant hosts.

To add "Pseudopositive reaction" to the results of serological test and modify this fig. to perform molecular test if pseudopositive reaction is observed. (See attached document: proposed amendment of Fig.1)

PPV widely infects not only woody plants but also herbaceous plants. So, sap inoculation for herbaceous plant hosts should be added.

Pseudopositive reaction may occur in serological test (ELISA, etc.) because PPV has a distant but serological relationship with PVY (Potato Virus Y) , Lycium spp., Solanum spp. which are common hosts to them. If pseudopositive reaction is detected, other test methods should be used for verification.

JAPAN

27





Editorial

SubstantiveRT-PCR or IC-RT-PCR(P1/P2 or 3NCR primers), Co-RT-PCR

Minimum requirements Recommended method for the detection and identification of Plum pox virus (e.g. during routine diagnosis of a pest widely established in a country)

“RT” is missing.

As above comment [12].

106 Figure 1 [13] Table

Para

Substantive

Substantive

Add dash line connecting from “Plum pox virus present” box (bottom left) to the Serological/Molecular tests box.

Minimum requirements for the detection and identification of Plum pox virus (e.g. during routine diagnosis of a pest widely established in a country). If no symptoms are observed on biological indicators, a serological or molecular test can be used. If positive results, a test should be run to back it up.

Because so many strains have been found that show other symptoms, or have different viruses showing similar symptoms, it should be strongly suggested that positive results are confirmed with molecular/serological tests.

When indexing PPV, very few times you will get a positive because the disease is not homogenous throughout the plant (i.e. plants may not show symptoms because virus is not present in all areas).

USA

USA


[14] Editorial In this diagnostic protocol, methods (including reference to brand names) are described as published, as these defined the original level of sensitivity, specificity and/or reproducibility achieved......

Methods still exist and hence still define the protocol

ANTIGUA AND BARBUDA, BARBADOS, CRWS, DOMINICA, SVG, SURINAME, TRINIDAD AND TOBAGO

108 3.Detection and [14] Substantive In this diagnostic protocol, methods See general comments ARGENTINA,

28





Identification (including reference to brand names) are described as published, as these defined the original level of sensitivity, specificity and/or reproducibility achieved. Use of names of chemicals or equipment in these diagnostic protocols implies no approval of them to the exclusion of others that may also be suitable. Laboratory procedures presented in the protocols may be adjusted to the standards of individual laboratories, provided that they are adequately validated.

Eliminado de acuerdo al comentario general realizado en referencia al uso de las marcas comerciales.

Removed according to general comment in reference to the use of brand names.


COSTA RICA

PERU, CUBA, PANAMA


[15]

Editorial

Substantive

In instances where the NPPO requires additional confidence in the identification of PPV (e.g. detection in an area where the virus is not known to occur), further tests may be done. In such cases where the initial identification was done using a molecular or serological method, subsequent tests should use a more sensitive technique than used in initial identification methods serological techniques and vice versa. Further tests may also be done to identify the strain of PPV present (Figure 1 2).

For better explanation

To confirm diagnosis, second test must be more sensitive than the first one.

Cambio editorial debido al comentario realizado en el párrafo 12.

Editorial change due to the comment made in paragraph 12.

ARGENTINA, BRAZIL, CHLE, COSAVE PARAGUAY, URUGUAY

COSTA RICA

PERU, CUBA, PANAMA


[15] Substantive In instances where the NPPO requires additional confidence in the identification of PPV (e.g. detection in an area where the virus is not known to occur or first finding in import/export), further tests may be done. Where the

To clarify critical cases where additional confidence is required

EU, EPPO

29





initial identification was done using a molecular method, subsequent tests should use serological techniques and vice versa. Further tests may also be done to identify the strain of PPV present (Figure 2).

111 3.1Biological detection and identification

[17] 1. Technical2. Editorial

Budwood to be grafted must be collected from at least three different branches of each plant (this is critical because of the uneven distribution of PPV).

1. For clarity of sampling method2. Less ambiguous statement

ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME, TRINIDAD AND TOBAGO, JAMAICA


[17]

Substantive

Budwood to be grafted must be collected from at least three different branches (this is critical because of the uneven distribution of PPV). The main indicator plants used for PPV indexing are seedlings of P. persicae cv. GF305, P. persicae × P. davidiana cv. Nemaguard, or P. tomentosa. Indicator plants are raised from seed, planted in a well-drained soil mixture and maintained in an insect-proof greenhouse between 18 °C and 25 °C until they are large enough to graft (usually 25–30 cm high with a diameter of 3–4 mm). The indicators must be graft-inoculated according to conventional methods such as bud grafting (Desvignes, 1999), using at least four replicates per indicator plant. For the inoculation, bud grafting and particularly chip budding are the most commonly used techniques. 3-4

To have more information to develop the technique correctly

ARGENTINA, BRAZIL, CHILE , COSAVE PARAGUAY, URUGUAY

30





Substantive

budwoods are collected from adult tree (fewer from younger tree), and two 10-15 mm stem pieces are collected on each budwood and randomly grafted on a single plant. These two stem pieces are grafted on opposite sides to each other 10-12 cm above the collar. Afterwards, appropriate grafting tape is used to hold and protect the grafted stem pieces. The upper stem piece has to be grafted just beneath an eye of the plant indicator. (Gentit, 2006).Following grafting the indicator plants are maintained in the same conditions and after 3 weeks are pruned to a few centimetres above the top graft (Gentit, 2006). Symptoms, in particular chlorotic banding and patterns, are observed on the new growth after 3–4 weeks and must be compared with positive and healthy controls. The symptoms in GF305 are observed in leaves with characteristic chlorotic patterns, yellow veins and yellowish spots. These symptoms are associated with leaf distortions and curling (Fig 1 y 2). In Prunus tomentosa the primary symptoms can be associated to chlorotic intervenial blotches in an oak leaf pattern. Chlorotic spots can also occur, these sometimes being necrotic with ageing of leaf. Leaf distortion, twisting and epinasty can also associated with PPV infection on P. tomentosa. (Gentit, 2006) (Fig 3, 4 and 5)

To have more information for to develop the technique correctly

31





Figure 1. GF305 PPV-D (photo: Damsteegt, V. D., Waterworth, H. E., Mink, G. I., Howell, W. E., and Levy. Plant Dis. 81:329-332)

32





Figure 2. GF305 (photo: Damsteegt, V. D., Scorza, R., Stone, A. L., Schneider, W. L., Webb, K., Demuth, M., and Gildow, F. E. Plant Dis. 91:18-23)

33





Figure 3 . Prunus tomentosa PPV-M (photo:Damsteegt, V. D., Waterworth, H. E., Mink, G. I., Howell, W. E., and Levy, L. Plant Dis. 81:329-332)

Figure 4 . Prunus tomentosa PPV-D (photo: Damsteegt, V. D., Waterworth, H. E., Mink, G. I., Howell, W. E., and Levy. Plant Dis. 81:329-332)

34





Figure 5 .Prunus tomentosa PPV-EA (photo:Damsteegt, V. D., Waterworth, H. E., Mink, G. I., Howell, W. E., and Levy. Plant Dis. 81:329-332).


[17] Sentence 4

Editorial Following grafting, the indicator plants are maintained in the same conditions and after 3 weeks are pruned to a few centimetres above the top graft (Gentit, 2006).

Add comma for clearer wording AUSTRALIA

114 3.1Biological detection and

[17] Sentence 5

Editorial Following grafting the The grafted indicator plants are maintained in at the

For clarity. JAMAICA

35





identification same conditions and, after 3 weeks, are pruned to a few centimetres above the top graft (Gentit, 2006).


[17] The main indicator plants used for PPV indexing are seedlings of P. persicae cv. GF305, P. persicae × P. davidiana cv. Nemaguard, or P. tomentosa

Substantive The main indicator plants used for PPV indexing are seedlings of P. persicae cv. GF 305, P. cerasifera cv. GF 31, P. tomentosa (for field and greenhouse indexing), or P. persicae × P. davidiana cv. Nemaguard, (in greenhouse).

The use of P. cerasifera cv. GF31 is not very common nevertheless it is possible to use. Could be included.

EU, EPPO


[17] New Sentence

Substantive

Substantive

To add new sentence after 3rd sentence: Seedlings grafted with indicator plant scions are also used.

To add new sentence after the last sentence: If symptoms are not observed in 5 weeks, the tested plant may be recognized as not infected. The instruments used for the tests should be disinfected (e.g. dunk in the 5% aqueous solution of Trisodium Phosphate for a few minutes). NPPO of the country where PPV has detected

It is difficult to secure the necessary number of seeds of a specific species. However, it is easy to secure a large volume of scions.

The period to judge if the tested plant is positive or negative should be noted. (In EPPO datasheet, symptoms are observed in 6-8 weeks.)Disinfection of the instruments and how to get the positive control are considered to be important for this protocol. And since PPV is transmissible by

JAPAN

36





should keep and maintain positive controls and distribute them if they are requested by the NPPO of the country where PPV has not occurred. In Biological indexing, be careful about the occurrence of aphids.

aphids, the occurrence of aphids during testing greatly reduces the reliability of the test result.


[17] Sentence 1

Sentence 6

Editorial

Editorial

Budwood to be grafted must be collected from at least three different branches (this is a critical step because of the uneven distribution of PPV).

The symptoms, in particular as chlorotic banding and patterns, are observed on the new growth after 3–4 weeks and must be compared with positive and healthy controls.



MEXICO


[17] As new sentence 6

Substantive PPV can also be detected in herbaceous indicator hosts by mechanical inoculation.

Clarity on non-reference to Herbaceous indexing. It would add value to add sentence that will cover Herbaceous indexing.

SOUTH AFRICA


[18] Editorial There is are no quantitative data published on the specificity, sensitivity or reliability of grafting.....

For sentence –verb agreement ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME, TRINIDAD AND TOBAGO, JAMAICA


[18]

Substantive

There is no quantitative data published on the specificity, sensitivity or reliability of grafting. The method is used widely in certification schemes and is considered to be a reliable and sensitive method of detection. However,

PPV symptoms can be confused with those of other graft transmissible agents

ARGENTINA, BRAZIL, CHILE, COSAVE PARAGUAY, URUGUAY,

37





Substantive

it is not a rapid test (symptom development requires several weeks post-inoculation), it requires dedicated facilities such as temperature-controlled greenhouse space, and the symptoms may be confused with those of other graft-transmissible agents. To confirm the presence of PPV another technique should be used.

Los síntomas de PPV pueden ser confundidos por otros agentes que se transmiten por injerto

A second test should be realized to confirm the presence of PPV because the symptoms can be confused with those of other graft transmissible agents.

Ver comentario anterior

PERU, CUBA, PANAMA

COSTA RICA


COSTA RICA


[18] New sentence at the end of the paragraph [18]

Substantive Moreover, there are some asymptomatic strains, which do not induce symptoms and thus are not detectable on indicator plants through biological detection (Figure 1).

To be consistent with figure 1 paragraph [13]

EU


[18] New sentence at the end of the paragraph [18]

Substantive Moreover, there are some asymptomatic strains, which do not induce symptoms and thus are not detectable on indicator plants through biological detection (Figure 1).

To be consistent with figure 1 paragraph [13]

EPPO to check, to provide further guidance.

EPPO


[18] Sentence 2

Editorial The method is used widely in certification schemes and is considered to be a reliable and sensitive method of detection.

Eliminate it to improve the sentence

MEXICO


[18] Sentence 3

Editorial However, it is not a rapid test (symptom development requires several weeks post-inoculation), as it requires dedicated facilities such as temperature-controlled greenhouse space, and the

For clarity. JAMAICA

38





symptoms observed may be confused with those of other graft-transmissible agents.

125 3.2Serological detection and identification

[20] Sentence 1

Editorial Enzyme-linked immunosorbent assays (ELISA) are highly recommended for screening large numbers of samples.

Acronym AUSTRALIA


[21] Editorial For sample processing, approximately 1 g of fresh plant material is weighed, cut into small pieces and placed in a suitable tube or plastic bag. About 20 volumes of extraction buffer are added and the sample is homogenized in tubes using a Polytron (Kinematica) or similar apparatus. Alternatively, the sample can be homogenized in plastic bags using a tissue homogenizer such as the Homex 6 machine (Bioreba) or a manual roller, hammer, or similar implement. The composition of the extraction buffer is phosphate-buffered saline (PBS) pH 7.2–7.4, containing 2% polyvinylpyrrolidone and 0.2% sodium diethyl dithiocarbamate (Cambra et al., 1994). Plant material should be homogenized thoroughly and used fresh.

Remove the deleted sentence and place as a footnote immediately after “…20 volumes of extraction buffer” in sentence 2 of this paragraph

ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, TRINIDAD AND TOBAGO SURINAME


[21]

Editorial

For sample processing, approximately 1 g of fresh plant material is weighed, cut into small pieces and placed in a suitable tube or plastic bag. About 20 volumes of extraction buffer are added and the sample is homogenized in tubes using an Polytron (Kinematica) homogenizer with rotor/stator system or similar apparatus. Alternatively, the sample can be homogenized in plastic bags using a tissue homogenizer such as

Commercial brands were removed instead; products are described, as agreed in relation with use of brand names in diagnostics protocols.


39





Substantive

an homogenizer with an electromechanical system to reduce the samples size, inside plastic bags, the Homex 6 machine (Bioreba) or a manual roller, hammer, or similar implement. As a recommendation the composition of the extraction buffer is should be phosphate-buffered saline (PBS) pH 7.2–7.4, containing 2% polyvinylpyrrolidone and 0.2% sodium diethyl dithiocarbamate (Cambra et al., 1994), or another recommended buffer. Plant material should be homogenized thoroughly and used fresh.

Can be more than one option of buffer



[21] For sample processing, approximately 1 g of fresh plant material is weighed, cut into small pieces and placed in a suitable tube or plastic bag. About 20 volumes of extraction buffer are added and the sample is homogenized in tubes using a Polytron (Kinematica) homogenizer with rotor/stator system or similar apparatus. Alternatively, the sample can be homogenized in plastic bags using a tissue homogenizer such as the Homex 6 machine (Bioreba) or a manual roller, hammer, or similar implement. The composition of the extraction buffer is phosphate-buffered saline (PBS) pH 7.2–7.4, containing 2% polyvinylpyrrolidone and 0.2% sodium diethyl dithiocarbamate (Cambra et al., 1994). Plant material should be homogenized thoroughly and used fresh.

A fin de eliminar la marca comercial y evitar que en el corto plazo se desactualice la norma.

Ver comentario anterior.

In order to remove brand names and prevent in a short-term outdating of the standard.

COSTA RICA

CUBA, PANAMA, PERU

40






[21] Sentences 1 and 2.

Substantive For sample processing, approximately 1 g 0.5 g of fresh plant material is weighed, cut into small pieces and placed in a suitable tube or plastic bag. About 20 Approximately 10 volumes of extraction buffer are added and the sample is homogenized in tubes using a Polytron (Kinematica) or similar apparatus.

Replace 1 g by 0.5g in sentence 1 and replace “About 20” in sentence 2 by “Approximately 10” because volumes of 1 g tissue with 20 ml buffer are impractical when using the standard bags for grinding samples for ELISA. These volumes will result in leakage over the top of the bags which may result in contamination. The Canadian PPV survey testing program uses 0.5 g of tissue in 5 mls of buffer to good effect. With the prudent preparation of samples these volumes are safer and adequate.

CANADA


[21] 2nd and 3rd sentences

Editorial About 20 volumes ml of extraction buffer are added and the sample is homogenized in tubes using a Polytron (Kinematica) or similar apparatus. Alternatively, the sample can be homogenized in plastic bags using a tissue homogenizer such as the Homex 6 machine (Bioreba) or a manual roller, hammer, or similar implements.

Technical clarity and correct English.

EU, EPPO


[21] Sentence 1

Editorial For sample processing, approximately 1 g of fresh plant material is weighed, cut into small pieces, and placed in a suitable tube or plastic bag.

A numerical value for weight is stated, so do not need the word ‘weighed’

JAMAICA


[21] Sentence 2

Editorial The sample is homogenized in About 20 volumes of extraction buffer are added and the sample is homogenized in tubes using a Polytron (Kinematica), Homex 6 machine (Bioreba) or similar apparatus apparati.

Use of too many words. JAMAICA

133 3.2Serological [21] Sentence Editorial Alternatively, the sample can be Removal of the entire sentence. JAMAICA

41





detection and identification

3 homogenized in plastic bags using a tissue homogenizer such as the Homex 6 machine (Bioreba) or a manual roller, hammer, or similar implement.

State only what you have or will use for homogenizing the material. Do not add alternatives. This is already understood.


[21] Sentence 3

Editorial Alternatively, the sample can be homogenized in plastic bags using a tissue homogenizer such as the Homex 6 machine (Bioreba) or a manual roller, hammer, or similar device apparatus.

This change improve the redaction

MEXICO


[22] Editorial 3.2.1 Double-antibody sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA)

Consistent formatting

For easy and sequential numbering

ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME, TRINIDAD AND TOBAGO



[23] Editorial DASI-ELISA, also called triple-antibody sandwich (TAS)-ELISA, should be performed according to Cambra et al. al. (1994) using the specific monoclonal antibody 5B-IVIA following the manufacturer’s instructions

Correct formatting ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME, TRINIDAD AND TOBAGO


[23] Substantive DASI-ELISA, also called triple-antibody sandwich (TAS)-ELISA, should be performed according to Cambra et al. (1994) using the an specific monoclonal antibody 5B-IVIA following the manufacturer’s instructions.

Although 5B IVIA is the only monoclonal antibody to PPV detection could be others antibodies available in the future


138 3.2Serological [24] Editorial ......The proportion of true negatives For consistency with the acronym ANTIGUA AND

42





detection and identification

(number of true negatives diagnosed by the technique/number of healthy plants) identified by DASI-ELISA using the 5B-IVIA monoclonal antibody was 99.0%, compared with real-time RT-PCR using purified nucleic acid (89.2%) or spotted samples (98.0%), or immunocapture IC- RT-PCR (96.1%). Capote et al. (2009) also reported that there is a 98.8% probability that a positive result obtained in winter with DASI-ELISA using the 5B-IVIA monoclonal antibody was a true positive.

that has already been defined BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME, TRINIDAD AND TOBAGO, JAMAICA


[24] Substantive

Substantive

5B-IVIA is currently the only monoclonal antibody that has been demonstrated to detect all strains of PPV, and does so with high reliability, specificity and sensitivity (Cambra et al., 2006a). In a DIAGPRO ring-test done by 17 laboratories using a panel of 10 samples (PPV-infected (PPV-D, PPV-M and PPV-D+M) and healthy) from France and Spain, DASI-ELISA using the 5B-IVIA monoclonal antibody was 95% accurate (number of true negatives and true positives diagnosed by the technique/number of samples tested). This accuracy was greater than that achieved with either immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) which was 82% accurate, or co-operational RT-PCR (Co-RT-PCR) which was 94% accurate (Cambra et al., 2006b; Olmos et al., 2007). The proportion of true negatives (number of

Could be others antibodies available in the future

For the purpose of this draft, it is not necessary the information in detail. The citation of the bibliographic reference is enough, in addition this detailed information is not specified for other techniques in this draft.

Esta información no resulta relevante a los fines del protocolo

This information is not relevant for the purposes of the Protocol


COSTA RICA

PANAMA, CUBA, PERU

43





true negatives diagnosed by the technique/number of healthy plants) identified by DASI-ELISA using the 5B-IVIA monoclonal antibody was 99.0%, compared with real-time RT-PCR using purified nucleic acid (89.2%) or spotted samples (98.0%), or immunocapture RT-PCR (96.1%). Capote et al. (2009) also reported that there is a 98.8% probability that a positive result obtained in winter with DASI-ELISA using the 5B-IVIA monoclonal antibody was a true positive.


[24] 1st sentence

Editorial 5B-IVIA is currently the only monoclonal antibody

Makes the statement more timely – the situation may change

EU, EPPO


[24] Editorial In a DIAGPRO ring-test done carried out by 17 laboratories using a panel of 10 samples, (PPV-infected (PPV-D, PPV-M and PPV-D+M) and healthy samples) from France and Spain, DASI-ELISA using the 5B-IVIA monoclonal antibody was 95% accurate (number of true negatives and true positives diagnosed by the technique/number of samples tested).

Improved wording and sentence structure

EU


[24] Editorial In a DIAGPRO ring-test done carried out by 17 laboratories using a panel of 10 samples, (PPV-infected (PPV-D, PPV-M and PPV-D+M) and healthy samples) from France and Spain, DASI-ELISA using the 5B-IVIA monoclonal antibody was 95% accurate (number of true negatives and true positives diagnosed by the technique/number of samples tested).

Improved wording and sentence structure

EPPO

44






[24] Sentence 1

Editorial 5B-IVIA is the only monoclonal antibody that has been demonstrated to detect all strains of PPV, and ….

Not necessary JAMAICA


[24] Sentence 1

Editorial 5B-IVIA is the only monoclonal antibody that has been demonstrated to detect all strains of PPV, and does so with high reliability, specificity and sensitivity (Cambra et al., 2006a).

This will improve the redaction MEXICO


[24] 2nd sentence

Substantive Monoclonal antibody accuracy is listed as 95%. Sensitivity should be listed. Where did this accuracy come from? In order to state sensitivity, we have to know how much is actually there. This pathogen is unevenly distributed. We should have to purify the virus first, then do a real solution series. Otherwise, we may rate false positive and false negatives.

USA


[25] Substantive DASI-ELISA kits based on the 5B-IVIA monoclonal antibody are available from AC Diagnostics, Inc. (Fayetteville, USA), Agritest (Valenzano, Italy), AMR Lab (Barcelona, Spain) and Real/Durviz (Valencia, Spain).

Commercial brands removed.See general comments

No corresponde citar marcas comerciales.

It is not the aim of the ISPMs to be the advertisements of traders; secondly, these sources may change in time.

Brand names were removed


COSTA RICA

EU, EPPO

CUBA, PANAMA, PERU

45






[25] Sentence 1

Substantive Delete the names of kits and companies in the parantheses.

This standards should not contain the brand names (commercial purpose)

CHINA


[25] Whole paragraph

editorial Move below paragraph 23 To link to “manufacturer’s instructions”

EPPO


[26] Editorial 3.2.2 Double-antibody sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA)

Consistency of formatting

For easy and sequential numbering referring to paragraph 22




[27] First sentence

Editorial The C conventional or biotin/streptavidin system of DAS-ELISA should be performed using kits based on the specific monoclonal antibody 5B-IVIA or on polyclonal antibodies that have been demonstrated to detect all strains of PPV without cross-reacting with other viruses or healthy plant material. The test should be done according to the manufacturer’s instructions.

For completeness of the sentence

No capitalization necessary


EU, EPPO


[27]

Substantive

Conventional or biotin/streptavidin system of DAS-ELISA should be performed using kits based on the specific monoclonal antibody 5B-IVIA or on polyclonal antibodies that have

To be consistent, see comments para 23

ARGENTINA, BRAZIL,

46





been demonstrated to detect all strains of PPV without cross-reacting with other viruses or healthy plant material. The test should be done according to the manufacturer’s instructions.

CHILE, COSAVE PARAGUAY, URUGUAY


[27] 1st sentence

Editorial antibodies that have been demonstrated to detect all strains of PPV without cross-reacting with other viruses or healthy plant material (Cambra et al., 2006a and Capote et al., 2009).

See the references Cambra et al., 2006a and Capote et al., 2009.

EU, EPPO


[28] Substantive Whereas the 5B-IVIA monoclonal antibody detects specifically, sensitively and reliably all PPV strains, some polyclonal antibodies ....




[28]

Editorial

Whereas the 5B-IVIA monoclonal antibody detects specifically, sensitively and reliably all PPV strains, some polyclonal antibodies are not specific and have limited sensitivity (Cambra et al., 1994; Cambra et al., 2006a). Therefore the use of additional methods is recommended in instances tests where polyclonal antibodies have been used and the NPPO requires additional confidence in the identification of PPV.

Para clarificar

To clarify

COSTA RICA

CUBA, PANAMA, PERU


[28] Sentence 2

Editorial Therefore the use of additional methods is recommended in assays instances where polyclonal antibodies have been used and the NPPO requires additional confidence in the identification of PPV.

This change improve the redaction

MEXICO


[28] 1st sentence

Editorial Whereas the 5B-IVIA monoclonal antibody detects specifically, sensitively and reliably detects all PPV strains…

Better wording EU, EPPO

47







Substantive DAS-ELISA kits (conventional or biotin/streptavidin system) for PPV detection are available from AC Diagnostics, Inc. (Fayetteville, USA), Adgen (Auchincruive, United Kingdom), Agdia Incorporated (Elkart, USA), Agritest (Valenzano, Italy), AMR Lab (Barcelona, Spain), Bio-Rad (Marnes La Coquette, France), Bioreba (Reinach, Switzerland), DSMZ (Braunschweig, Germany), Hortitech (Selby, United Kingdom), Ingenasa (Madrid, Spain), Loewe Biochemica (Sauerlach, Germany), Plant Research International (Wageningen, the Netherlands), and Real/Durviz (Valencia, Spain).

Removed because are all commercial brands.See general comments




To delete all sentence because is not part of the test, is complimentary information because we are referring only brand names and where we can find it.


COSTA RICA

EU, EPPO

PANAMA, CUBA, PERU

MEXICO


[29] Substantive Delete the names of kits and companies in the parantheses.

This standards should not contain the brand names (commercial purpose)

CHINA

159 3.3 Molecular detection and identification

[31] Add new first para

Add last sentence

Substantive

Substantive

At the time of writing, these are the most up to date protocols recommended for molecular detection and identification. Literature must be consulted to make sure the most accurate, up to date method is used. NPPOs may use one or another, and not necessarily all methods listed.

Nevertheless, conventional PCR can be used when it is the only method

Disclaimer should be added because methods are always changing. New, better antibodies are being developed, so NPPOs should understand that the science behind the protocol is always evolving.It should be made clear to NPPOs that they do not need to use all methods listed.

The protocol is supposed to be applicable to all member

USA

USA

48





available. countries. Some countries may only have conventional PCR method. Many labs do visual and graft inoculation because they do not have the materials, technology, etc., necessary to do some higher tech options.


[32] Technical Fresh or frozen (stored between −20 °C and −80 °C for periods of at least up to one year) plant extracts can be used for molecular tests. .....

This is the correct time-frame ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME, TRINIDAD AND TOBAGO, JAMAICA


[32] 1st sentence

Technical Fresh or frozen (stored between −20 °C and −80 °C for periods of at least no more than one year) plant extracts can be used for molecular tests.

Correction of the text. EU, EPPO


[32]

Substantive

Fresh or frozen (stored between −20 °C and −80 °C for periods of at least one year) plant extracts can be used for molecular tests. With the exception of IC-RT-PCR (for which RNA isolation is not required), RNA extraction should be done using appropriately validated protocols the RNeasy Plant Minikit (Qiagen), the UltraClean™ Plant RNA Isolation Kit (Mo Bio) or other appropriately validated protocols, according to the manufacturer’s instructions. Alternatively for real-time RT-PCR, spotted plant extracts (5 μl) or printed tissue sections can be immobilized on Whatman 3MM an specific paper or nylon membranes and

Commercial brands removed.See general comments.


49





analysed by real-time RT-PCR (Olmos et al., 2005; Osman and Rowhani, 2006; Capote et al., 2009). It is not recommended to use spotted or tissue-printed samples in conventional PCR because of the lesser sensitivity compared with real-time RT-PCR.


[32] Fresh or frozen (stored between −20 °C and −80 °C for periods of at least one year) plant extracts can be used for molecular tests. With the exception of IC-RT-PCR (for which RNA isolation is not required), RNA extraction should be done using the RNeasy Plant Minikit (Qiagen), the UltraClean™ Plant RNA Isolation Kit (Mo Bio) or other appropriately validated protocols, according to the manufacturer’s instructions. Alternatively for real-time RT-PCR, spotted plant extracts (5 μl) or printed tissue sections can be immobilized on Whatman 3MM a specific paper or nylon membranes and analysed by real-time RT-PCR (Olmos et al., 2005; Osman and Rowhani, 2006; Capote et al., 2009). It is not recommended to use spotted or tissue-printed samples in conventional PCR because of the lesser sensitivity compared with real-time RT-PCR.



COSTA RICA

CUBA, PANAMA, PERU


[32] 3rd-4th row

Editorial With the exception of IC-RT-PCR (for which RNA isolation is not required), RNA extraction should be done using the RNeasy Plant Minikit (Qiagen), the UltraClean™ Plant RNA Isolation Kit (Mo Bio) or other appropriately validated protocols, according to the


EU, EPPO

50





manufacturer’s instructions.


[32] Last sentence

Editorial It is not recommended to use spotted or tissue-printed samples in conventional PCR because of the lesser lower sensitivity compared with real-time RT-PCR.

Better wording EU, EPPO


[32] Lines 1 and 2

Line 6

Substantive ... one year) plant extracts, prepared on individual plastic bags, can be used for……(5 μl) or printed tissue sections or squashes of plant material can be immobilized…

Extracts for PPV detection by molecular methods must be prepared in plastic bags to avoid contamination among samples.

Another possibility already described in the literature.

EU, EPPO


[32] As new paragraph 33

Substantive To increase sensitivity, one would need to design a nested PCR reaction and the same primers mentioned could still be used.

A nested PCR should be considered, but needs to be developed

SOUTH AFRICA


[32] First sentence

Substantive delete The protocol states that frozen ítems should be processed within one year. This makes it seem like it would be an acceptable thing to do but in reality, samples should be sampled as soon as possible. Frozen material should only be used as a last ditch effort. Test results would be hard to believe if negative because frozen material can contribute to increases in false negatives. The protocol without this sentence, would be more consistent with para 11, where it states that samples “can be stored at 4°C for not more than 7 days before processing.”

USA

169 3.3.1Reverse transcription-

[33]Substantive

3.3.1 One step Rreverse transcription-polymerase chain reaction

To clarify that it is a reaction of one step

ARGENTINA, BRAZIL,

51





polymerase chain reaction (RT-PCR)

(One step RT-PCR)

Para clarificar que se trata de una reacción de un paso.

CHILE, COSAVE PARAGUAY, LARWS, PERU, URUGUAY

COSTA RICA

170 3.3.1Reverse transcription-polymerase chain reaction (RT-PCR)

[34] Add first sentence

Substantive Use of these primers has been successful at trapping the PPV virus. Primers are universally used.

Suggest to add more information about each primer mentioned in the text.

USA


[35] 1. Editorial

2. Editorial

The 25 μl reaction mixture is composed as follows: 1 μM of each primer (P1/P2 or the 3′NCR primer pair), 250 μM dNTPs, 1 unit AMV reverse transcriptase, 0.5 units Taq DNA polymerase, 2.5 μl 10 × Taq polymerase buffer, 1.5 mM MgCl22, and 0.3% Triton X-100. The reaction is performed under the following thermocycling conditions: 45 min at 42 °C, 2 min at 94 ºC, 40 cycles of 30 s at 94 °C, 30 s at either 60 °C (P1/P2 primers) or 62 °C (3′NCR primers), 1 min at 72 °C, followed by a final extension for 10 min at 72 °C and then quickly cooled to room temperature. The PCR products are analysed by gel electrophoresis. The P1/P2 and 3′NCR primers produce a 243 base pair (bp) and 220 bp amplicon, respectively.

1. Correct way of writing the formula

Formatting corrections

2. Added for clarity

ANTIGUA AND BARBUDA, , BARBADOS, DOMINICA, SVG, SURINAME, JAMAICA


[35] Sentence 1 Editorial The 25 μl reaction mixture is composed as follows: 1 μM of each primer (P1/P2 or the 3′NCR primer pair), 250 μM dNTPs, 1 unit AMV reverse transcriptase, 0.5 units Taq DNA

Correct way of writing the formula.

AUSTRALIA, EU, EPPO, TRINIDAD AND TOBAGO, THAILAND, SOUTH

52





polymerase, 2.5 μl 10 × Taq polymerase buffer, 1.5 mM MgCl22, and 0.3% Triton X-100.

AFRICA


[35] Sentence 2 Editorial The reaction is performed under the following thermocycling conditions: 45 min at 42 °C, 2 min at 94 ºC, 40 cycles of 30 s at 94 °C, 30 s at either 60 °C (P1/P2 primers) or 62 °C (3′NCR primers), 1 min at 72 °C, followed by a final extension for 10 min at 72 °C and then quickly cooled to room temperature.

Added for clarity. TRINIDAD AND TOBAGO


[35]

Editorial

The 25 μl reaction mixture is composed as follows: 1 μM of each primer (P1/P2 or the 3′NCR primer pair), 250 μM dNTPs, 1 unit AMV reverse transcriptase, 0.5 units Taq DNA polymerase, 2.5 μl 10 × Taq polymerase buffer, 1.5 mM MgCl2, and 0.3% Triton X-100 and add 300 pg to 2 μg of RNA or DNA (specific detection and quantification of Plum Pox Virus by real time fluorescent reverse transcription PCR (Schneider et al 2004)). The reaction is performed under the following thermocycling conditions: ...

Text was added to clarify ARGENTINA, BRAZIL, CHILE, COSAVE PARAGUAY, URUGUAY


[35] The 25 μl reaction mixture is composed as follows: 1 μM of each primer (P1/P2 or the 3′NCR primer pair), 250 μM dNTPs, 1 unit AMV reverse transcriptase, 0.5 units Taq DNA polymerase, 2.5 μl 10 × Taq polymerase buffer, 1.5 mM MgCl2, and 0.3% Triton X-100. The reaction is performed under the following thermocycling

Ver comentarios generales. COSTA RICA

53





conditions: 45 min at 42 °C, 2 min at 94 ºC, 40 cycles of 30 s at 94 °C, 30 s at either 60 °C (P1/P2 primers) or 62 °C (3′NCR primers), 1 min at 72 °C, followed by a final extension for 10 min at 72 °C and quickly cooled to room temperature. The PCR products are analysed by gel electrophoresis. The P1/P2 and 3′NCR primers produce a 243 base pair (bp) and 220 bp amplicon, respectively.


[35] Sentence 1

Editorial The 25 μl reaction mixture is composed as follows: 1 μM of each primer (P1/P2 or the 3′NCR primer pair), 250 μM dNTPs, ……

Should explain the acronym NCR (non-coding region) before being use or mention in this paragraph.

JAMAICA


[35] Sentence 2

Editorial …..62 °C (3′NCR primers), and 1 min at 72 °C,…..

‘and’ indicates the last input in the cycles

JAMAICA


[35] 2nd Sentence

Substantive …1 min at 72 °C, followed by a final extension for 10 min at 72 °C and quickly cooled to room temperature 4 °C.

Many journal articles generally state that the final temperature of PCR is 4oC.

JAPAN


[35] Sentence 1

Sentence 2

Substantive

Substantive

The 25 μl reaction mixture is composed as follows: 1 μM of each primer (P1/P2 or the 3′NCR primer pair), 250 μM dNTPs, 1 unit AMV reverse transcriptase, 0.5 units Taq DNA polymerase, 2.5 μl 10 × Taq polymerase buffer and 1.5 mM MgCl2, and 0.3% Triton X-100.

The reaction is performed under the following thermocycling conditions: 45 min at 42 °C, 2 min at 94 ºC, 40 cycles of 30 s at 94 °C, 30 s at either 60 °C

- Specify how is generated the first Chaín with oligos dT and next how the second chain with reverse transcriptase. -Specify the concentration of RNA. -Eliminate the triton X-100 because is not necessary in the reaction.

The thermocycling conditions of generation of first chain (with

MEXICO

54





(P1/P2 primers) or 62 °C (3′NCR primers), 1 min at 72 °C, followed by a final extension for 10 min at 72 °C and quickly cooled to room temperature.

oligos dT) and generation of second chain with reverse transcriptase are mixed. They must be separated.


[35] The 25 μl reaction mixture is composed as follows: 1 μM of each primer (P1/P2 or the 3′NCR primer pair), 250 μM dNTPs, 1 unit AMV reverse transcriptase, 0.5 units Taq DNA polymerase, 2.5 μl 10 × Taq polymerase buffer, 1.5 mM MgCl2, and 0.3% Triton X-100 and 5 µl of RNA . The reaction is performed under the following thermocycling conditions: 45 min at 42 °C, 2 min at 94 ºC, 40 cycles of 30 s at 94 °C, 30 s at either 60 °C (P1/P2 primers) or 62 °C (3′NCR primers), 1 min at 72 °C, followed by a final extension for 10 min at 72 °C and quickly cooled to room temperature. The PCR products are analysed by gel electrophoresis. The P1/P2 and 3′NCR primers produce a 243 base pair (bp) and 220 bp amplicon, respectively.

To complete the reaction mixture(it is necessary to add in the phrase “ and 5 µl of RNA” which represents the sample to be tested ).All the text in the standard has included the sample. This must be forgotten.

PERÚ


[36] Sentence 2

Editorial ....The assay was able to detect 20 fg of viral RNA, corresponding to 2000 2 000 viral particles (Wetzel et al., 1991).....

For consistency with the way other figures are written throughout the document.

Add a blank (2 000 insead of 2000)


EU, EPPO

182 3.3.1Reverse [36] Sentence Substantive The assay was able to detect 20 10 fg The paper specified 10 fg JAMAICA

55





transcription-polymerase chain reaction (RT-PCR)

2 of viral RNA, corresponding to 2000 viral particles (Wetzel et al., 1991).

corresponding to 2000 viral particles, not 20 fg.


[36] Substantive The method of Wetzel et al. (1991) was evaluated by testing PPV isolates from Mediterranean areas (Cyprus, Egypt, France, Greece, Spain and Turkey). The assay was able to detect 20 fg of viral RNA, corresponding to 2000 viral particles (Wetzel et al., 1991). The method, designed by Levy and Hadidi (1994), was evaluated using PPV isolates from Egypt, France, Germany, Greece, Hungary, Italy, Spain and Romania.

For the purpose of this draft, it is not necessary the information in detail.

Para ser consistente con lo eliminado en el párrafo 24.

This information is not relevant for the purposes of the Protocol.


COSTA RICA

LARWS, PERU


[36] Sentence 3

editorial The method of designed by Levy and Hadidi (1994), was evaluated using PPV isolates from Egypt, France, Germany, Greece, Hungary, Italy, Spain and Romania.

To improve the redaction MEXICO

185 1.3 Production factors that affect risk

[37] Editorial 3.3.2 Immunocapture Polymerase Reverse Transcription Polymerase Chain Reaction (IC-RT-PCR)

For consistency ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME, TRINIDAD AND TOBAGO, JAMAICA

186 1.3 Production factors that affect risk

[37] Editorial " Immunocapture RT-PCR” should be corrected to” Immunocapture RT-PCR (IC-RT-PCR)."

"(IC-RT-PCR)" is missing.

For consistency with paragraph 40, sentence 1.

JAPAN


187 3.3.2 Immunocapture RT-PCR

[38] Line 2 Substantive ... extracted as in section 3.2. on plastic bags to avoid contamination.

As in paragraph 32. EU, EPPO

56






[39] Editorial ....Wash the tubes three times with 150 μl of sterile PBS-Tween (washing buffer).....

Redundant ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME, TRINIDAD AND TOBAGO, JAMAICA


[39]

Substantive

Editorial

Prepare a dilution (1 μg ml-1) of polyclonal antibodies or PPV-specific monoclonal antibody (5B-IVIA) in carbonate buffer pH 9.6. Add 100 μl of the diluted antibodies into microfuge tubes and incubate at 37 °C for 3 h. Wash the tubes twice with 150 μl of sterile PBS-Tween (washing buffer). Clarify 100 μl of plant extract (see section 3.2) by centrifugation (5 min at 15 500 × g), and add the supernatant to the coated microfuge tubes. Incubate for 2 h on ice or at 37 °C (Rosner et al , 1998). Wash the tubes three times with 150 μl of sterile PBS-Tween (washing buffer).


Relevant reference was added.



[39] After Sentence 3

Substantive Add sentence:Rinse with water Rnase free for 3 minutes.

To discard buffer salt.

EU, EPPO


[40]

Substantive

Inherent problems in IC-RT-PCR (inhibition and false positives due to contamination) make this method less reliable than other recommended serological and molecular tests. IC-RT-PCR using the 5B-IVIA monoclonal antibody has been validated in a DIAGPRO ring-test showing an accuracy of 82% for PPV detection

Removed because it is not relevant information for the purpose of this Draft

ARGENTINA, BRAZIL, CHILE, COSAVE PARAGUAY,

57





Substantive(Cambra et al., 2006b; Olmos et al., 2007). The method also requires the use of specific antibodies and can be time consuming. Capote et al. (2009) reported that there is a 95.8% probability that a positive result obtained in winter with IC-RT-PCR using the 5B-IVIA monoclonal antibody was a true positive.





URUGUAY

COSTA RICA


COSTA RICA

PANAMA, CUBA, PERU



Substantive Inherent problems in IC-RT-PCR (inhibition and false positives due to contamination) make this method less reliable than other recommended serological and molecular tests. IC-RT-PCR using the 5B-IVIA monoclonal antibody has been validated in a DIAGPRO ring-test showing an accuracy of 82% for PPV detection (Cambra et al., 2006b; Olmos et al., 2007). The method also requires the use of specific antibodies and can be time consuming. Capote et al. (2009) reported that there is a 95.8% probability that a positive result obtained in winter with IC-RT-PCR using the 5B-IVIA monoclonal antibody was a true positive.IC-RT-PCR is more sensitive and reliable than standard serological and

The paragraph contains inaccurate statements and should be replaced by a new paragraph. IC-RT-PCR is more sensitive and reliable than other serological and molecular tests (Candresse et al., 1994. EPPO Bulletin, 24:585-594). It is possible that the use of a monoclonal antibody might have limited sensitivity in the DIAGPRO ring-test. Polyclonal antibodies are usually better suited to this assay. Sentence 3 is incorrect. IC-RT-PCR does not require RNA extraction which reduces cost and time. Also sentences #2 and #4 are contradictory.

CANADA

58





molecular tests (Candresse et al., 1994. EPPO Bulletin, 24:585-594). IC-RT-PCR using the 5B-IVIA monoclonal antibody has been validated in a DIAGPRO ring-test showing an accuracy of 82% for PPV detection (Cambra et al. 2006b; Olmos et al. 2007). The method requires the use of specific antibodies; however direct-binding methods eliminate the need for any antibody (James, 1999. J. Virol. Methods 83: 1-9; Rowhani et al., 1995. Phytopathol. 85: 347-352). IC-RT-PCR and direct-binding RT-PCR do not require RNA extraction which reduces cost, time, and complexity. Capote et al. (2009) reported that there is a 95.8% probability that a positive result obtained in winter with IC-RT-PCR using the 5B-IVIA monoclonal antibody was a true positive.

193 3.3.3Co-operational RT-PCR

[41] Editorial 3.3.3 Co-operational Reverse Transcription - Polymerase Chain Reaction (Co - RT-PCR)

For consistency ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME, TRINIDAD AND TOBAGO, JAMAICA


[41] Editorial "Co-operational RT-PCR” should be corrected to “Co-operational RT-PCR (CO-RT-PCR)."

"(CO-RT-PCR)" is missing.

For consistence with paragraph 42.

JAPAN



[42] Para Substantive Suggest to add information about each primer mentioned in the text.

USA

59






[43] 1. Editorial2. Editorial

The 20 μl reaction mixture is composed as follows: 0.1 μM of P1 and P2 primers, 0.05 μM of P10 and P20 primers, 400 μM dNTPs, 2 units AMV reverse transcriptase, 1 unit Taq DNA polymerase, 2 μl 10 × reaction buffer, 3 mM MgCl22, 5% DMSO, and 0.3% Triton X-100. The reaction mixture and 5 μl of RNA sample are added to a sterile microfuge tube. The RT-PCR is performed under the following thermocycling conditions: 45 min at 42 °C, 2 min at 94 °C, 60 cycles of 15 s at 94 °C, 15 s at 50 °C, 30 s at 72 °C, followed by a final extension for 10 min at 72 °C and quickly cooled to room temperature.

1. Care to be taken in the final version of the draft that the 10 does not fall on a separate line to the x

2. Correct formula format

ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SURINAME, TRINIDAD AND TOBAGO


[43] Line 3 Editorial MgCl22 Formatting correction EU, EPPO, SOUTH AFRICA, SVG, THAILAND, TRINIDAD AND TOBAGO



Substantive - The authors should indicate the specific reagent volumes for ease of replication and implementation. Some volumes are given but not all. The reagent volumes and steps involved in co-operational RT-PCR are not clear. It is not clear if the final reaction volume is 20 or 25 ul. Text should be modified for better clarity of this paragraph.

CANADA


[43] Sentence 2

Substantive The reaction mixture and 5 μl of RNA sample are added to a sterile microfuge PCR tube.

There is a difference between microcentrifuge tubes and PCR tubes

JAMAICA

60






[43] Sentence 3

Editorial ….. 60 cycles of 15 s at 94 °C, 15 s at 50 °C, and 30 s at 72 °C, followed by a final extension for 10 min at 72 °C, and then quickly cooled to room temperature.

‘and’ indicates the last input in the cycle

JAMAICA


[43] Sentence 1

Sentence 3

Substantive

Substantive

The 20 μl reaction mixture is composed as follows: 0.1 μM of P1 and P2 primers, 0.05 μM of P10 and P20 primers, 400 μM dNTPs, 2 units AMV reverse transcriptase, 1 unit Taq DNA polymerase, 2 μl 10 × reaction buffer, 3 mM MgCl2, 5% DMSO, and 0.3% Triton X-100. The reaction mixture and 5 μl of RNA sample are added to a sterile microfuge tube.

The RT-PCR is performed under the following thermocycling conditions: 45 min at 42 °C, 2 min at 94 °C, 60 cycles of 15 s at 94 °C, 15 s at 50 °C, 30 s at 72 °C, followed by a final extension for 10 min at 72 °C and quickly cooled to room temperature.

- Specify how is generated the first Chaín with oligos dT and next how the second chain with reverse transcriptase. -Specify the concentration of RNA.

The thermocycling conditions to generate the first chain (with oligos dT) and to generate the second chain with reverse transcriptase are mixed. They must be separated.

MEXICO


[44] 1. Editorial2. Editorial

The RT-PCR reaction is coupled to a colorimetric detection of amplicons using a 3´digoxigenin-labelled PPV universal probe (5′-TCG TTT ATT TGG CTT GGA TGG AA-Digoxigenin-3′; Roche Molecular Biochemicals) as follows.: D denature the amplified cDNA at 95 °C for 5 min and immediately place on ice. .....

1. Correct punctuation2. letter case change

ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME, TRINIDAD AND TOBAGO


[44]

Substantive

The RT-PCR reaction is coupled to a colorimetric detection of amplicons using a 3´digoxigenin-labelled PPV universal probe (5′-TCG TTT ATT TGG CTT GGA TGG AA- Commercial brand removed. ARGENTINA,

61





Substantive

Digoxigenin-3′; Roche Molecular Biochemicals) as follows. ............................. ..................... The substrate solution is prepared by mixing 45 μl NBT solution (75 mg ml-1 nitro blue tetrazolium salt in 70% (v/v) dimethylformamide) and 35 μl BCIP solution (50 mg ml-1 5-bromo-4chloro-3indolyl phosphate toluidinium salt in 100% dimethylformamide) in 10 ml of detection buffer; alternatively, dilute a substrate tablet according to the manufacturer's instructions (included in the Roche Multicolor Detection Kit) in 10-ml detection buffer. After 1 h incubation with the substrate stop the reaction by washing with water.

See general comments.

No corresponde citar marcas comerciales


Commercial brand removed and text added as appropriated.See general comments.

No corresponde citar marcas comerciales Costa Rica y se agregó texto para clarificar.

Brand names were removed and text has been added to clarify


COSTA RICA

CUBA, PANAMA, PERU


COSTA RICA

CUBAM PANAMA , PERU


[44] - Is this paragraph proposing using the Roche Multicolor Detection Kit? If yes, state so at the beginning of the paragraph. If not, the reader needs to know what is in the blocking and detection buffers.

JAMAICA


[45] Substantive This method was 100 times more sensitive than RT-PCR using the assay of Wetzel et al. (1991) (Olmos et al.,


ARGENTINA, BRAZIL, CHILE,

62





2002). The method was validated in the DIAGPRO ring-test and had an accuracy of 94% (Cambra et al., 2006b; Olmos et al., 2007). Para ser consistente con lo

eliminado en el párrafo 24.


COSAVE PARAGUAY, URUGUAY

COSTA RICA

PANAMA, CUBA, PERU



Substantive - The authors should provide further justification as to the merits of this assay apart from being 100 times more sensitive than RT-PCR. IC-RT-PCR has a similar level of sensitivity and is simpler to implement. This assay is more complex and time consuming than IC-RT-PCR. IC-RT-PCR is also 100 times more sensitive than RT-PCR (Candresse et al., 1994). The value of this assay has not been adequately described.

CANADA

207 3.3.4Real-time RT-PCR Mitigation Measures

[48] 1& 2. Editorial3 Editorial

The 25 μl reaction mixture is composed as follows: 1 × reaction mix (0.2mM of each dNTP and 1.2 mM MgSO44), 200 nM of forward and reverse primers, 100 nM TAMRA probe, 4.8 mM MgSO44 and 0.5 μl RT/Platinum® Taq mix (Superscript™ One-Step RT-PCR with Platinum® Taq kit [(Invitrogen)]).

1. & 2. Correct formula format

3. better for the parentheses to be of different forms in such instances.

ANTIGUA AND BARBUDA, AUSTRALIA, BARBADOS, DOMINICA, SOUTH AFRICA, SVG, SURINAME, THAILAND, TRINIDAD AND TOBAGO


[48] The 25 μl reaction mixture is composed as follows: 1 × reaction mix (0.2mM of each dNTP and 1.2 mM MgSO4), 200

63





Substantive

nM of forward and reverse primers, 100 nM TAMRA probe, 4.8 mM MgSO4 and 0.5 μl RT/Platinum® Taq mix (Superscript™ One-Step RT-PCR with Platinum® Taq kit (Invitrogen1). The reaction mixture and 300 pg of RNA template are added to a sterile microfuge tube or equivalent. ...

1 The use of the brand Invitrogen for the Superscript™ one step RT-PCR and the Platinum® Taq kit in this diagnostic protocol implies no approval of them to the exclusion of others that may also be suitable. This information is given for the convenience of users of this protocol and does not constitute an endorsement by the CPM of the chemical, reagent and/or equipment named. Equivalent products may be used if they can be shown to lead to the same results.

Footnote added in relation to the use of brand names. See general comments




Substantive - The authors should indicate the specific reagent volumes for ease of replication and implementation. It is not clear if the final reaction volume is 25 ul or 30 ul. IN sentence 2, the statement “The reaction mixture and 300 pg of RNA template...” is not clear or precise. The text should be modified for better clarity.

CANADA


[48] The 25 μl reaction mixture is composed as follows: 1 × reaction mix (0.2mM of each dNTP and 1.2 mM MgSO4), 200 nM of forward and reverse primers, 100 nM TAMRA probe, 4.8 mM MgSO4

Ver comentario general en relación a las citas de las marcas comerciales.

See general comments regarding

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and 0.5 μl RT/Platinum® Taq mix (Superscript™ One-Step RT-PCR with Platinum® Taq kit (Invitrogen)). The reaction mixture and 300 pg of RNA template are added to a sterile microfuge tube or equivalent. The RT-PCR is performed under the following thermocycling conditions: 15 min at 52 °C, 5 min at 95 °C, 60 cycles of 15 s at 95 °C, 30 s at 60 °C, and quickly cooled to room temperature. The PCR products are analysed in real-time according to the manufacturer’s instructions.

the use of brand names. PANAMA, PERU


[48] Sentence 3

Substantive The RT-PCR is performed under the following thermocycling conditions: 15 min at 52 °C, 5 min at 95 °C, 60 cycles of 15 s at 95 °C, 30 s at 60 °C, and quickly cooled to room temperature. The PCR products are analysed in real-time according to the manufacturer’s instructions.

The thermocycling conditions of generation of first chain (with oligos dT) and thermocycling conditions with reverse transcriptase are mixed. And60 cycles are too many and the result cannot be trusted. they must be separated.

MEXICO


[48] Line 1

Line 2

Editorial 0.2mM 0.2 mM

MgSO44 TAMRA TaqMan

Space between: 0.2 mM

Twice in the same line MgSO44

EU, EPPO


[49] Substantive The method of Schneider et al. (2004) was evaluated by testing PPV isolates from the United States, strains PPV-C, PPV-D, PPV-EA and PPV-M, and eight other viral species. The method was specific and able to detect consistently 10–20 fg of viral RNA (Schneider et al., 2004). The method could also detect PPV in a number of hosts and in the leaves, stems, buds and roots of P. persica.


Este párrafo no aporta información relevante para el protocolo.



COSTA RICA

CUBA,

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PANAMA, PERU


[49] Sentence 2

Editorial Need to clarify what “fg” stand for Clarity SEYCHELLES


[50] After paragraph 50

Substantive Add new paragraph after 50:It could be used primers and probe of an internal control for real-time RT-PCR protocol.

The internal control may be included to ensure the correct performance of the assay.

The TAQMAN reactions should included the internal control to give a level of confidence to the diagnostic, same as the real time reactions using SYBR green as pointed in the text of this protocol.

PERU


[51]

Substantive

The 25 μl reaction mixture is composed as follows: 1 μM of P241 primer, 0.5 μM each of P316D and P316M primers, 200 nM TAMRA probe, 1 × TaqMan Universal PCR Master Mix (Applied Biosystems2), and 1 × MultiScribe and RNase Inhibitor Mix (Applied Biosystems3) The reaction mixture and 5 μl of RNA template are added to a sterile microfuge tube or equivalent. The...2, 3 The use of the brand Applied Biosystems for TaqMan Universal PCR Master Mix, and for MultiScribe and RNase Inhibitor Mix in this diagnostic protocol implies no approval of them to the exclusion of others that may also be suitable. This information is given for the convenience of users of this protocol and does not constitute an endorsement by the CPM of the



66





chemical, reagent and/or equipment named. Equivalent products may be used if they can be shown to lead to the same results.


[51] The 25 μl reaction mixture is composed as follows: 1 μM of P241 primer, 0.5 μM each of P316D and P316M primers, 200 nM TAMRA probe, 1 × TaqMan Universal PCR Master Mix (Applied Biosystems), and 1 × MultiScribe and RNase Inhibitor Mix (Applied Biosystems). The reaction mixture and 5 μl of RNA template are added to a sterile microfuge tube or equivalent. The RT-PCR is performed under the following thermocycling conditions: 30 min at 48 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 60 s at 60 °C, and quickly cooled to room temperature. The PCR products are analysed in real-time according to the manufacturer’s instructions.


See general comments regarding the use of brand names.

COSTA RICA

CUBA, PANAMA, PERU


[51] Sentence 2

Sentence 3

Substantive

Substantive

The reaction mixture and 5 μl of RNA template are added to a sterile microfuge tube or equivalent.

The RT-PCR is performed under the following thermocycling conditions: 30 min at 48 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 60 s at 60 °C, and quickly cooled to room temperature. The PCR products are analysed in real-time according to the manufacturer’s instructions.

-The concentration of RNA must be specificated.

-Check the thermocycling conditions

MEXICO



Substantive - The final volumes and reagent volumes are not clearly described. It is not clear if the final reaction

CANADA

67





volume is 25 ul or 30 ul. The authors should indicate the specific reagent volumes for ease of replication and implementation.


[51] Line 2 Editorial TAMRA TaqMan Term normally used EU, EPPO


[51] Substantive The reaction mixture and 5 μl of RNA sample are added to a sterile microfuge PCR tube or equivalent.

There is a difference between microcentrifuge tubes and PCR tubes. PCR tubes are used for RT and PCR by design. Be specific.

JAMAICA


[51] 3rd Sentence

Substantive ..., 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 60 s at 60 °C, and quickly cooled to room temperature 4 ºC.

Many journal articles state that the reaction mixture should be stored at 4oC.

JAPAN


[52] Substantive The method of Olmos et al. (2005) was evaluated using three isolates each of PPV-D and PPV-M, and was 1 000 times more sensitive than DASI-ELISA using the 5B-IVIA monoclonal antibody. The proportion of true positives (number of true positives diagnosed by the technique/number of healthy plants) identified correctly by real-time RT-PCR using TaqMan (Olmos et al., 2005) and purified nucleic acid was 97.5%, compared with real-time RT-PCR using spotted samples (93.6%), immunocapture RT-PCR (91.5%) or DASI-ELISA using the 5B-IVIA monoclonal antibody (86.6%) (Capote et al., 2009).





COSTA RICA

CUBA, PANAMA, PERU


[52] Line 3 Technical ... true positives (number of true positives diagnosed by the technique/number of PPV infected plants healthy) identified…

It was a mistake in the manuscript (number of true positives diagnosed by the technique/number of healthy

EU, EPPO

68





plants) would be FALSE positives.


[55]

Editorial

A two-step RT-PCR protocol is used. The RT reaction is composed as follows: 2 μl of 10 μM P1 primer, 2 μl of 10 μM Nad5-R primer, 4 μg total RNA and 5 μl water. Incubate 72 °C for 5 min, place on ice. Add 4 μl 5 × first strand buffer (Invitrogen4), 2 μl 0.1 M DTT, 1 μl 10 mM dNTPs, 0.5 μl RNaseOUTTMTM (40 units μl−1) (Invitrogen5), 1 μl Superscript™ II (Invitrogen6) and 2.5 μl water. Incubate 42 °C for 60 min followed by 99 °C for 5 min. The 25 μl PCR reaction mixture is composed as follows: 400 nM PPV-U primer, 350 nM PPV-FM primer, 150 nM PPV-FD primer, 200 nM PPV-RR primer, 100 nM Nad5-F primer, 100 nM Nad5-R primer, 200 μM dNTPs, 2mM MgCl2, 1 × Karsai buffer (Karsai et al., 2002), 1:42 000 SYBR Green I (Sigma7) and 0.1 μl Platinum® Taq DNA high fidelity polymerase (Invitrogen8). The reaction mixture and 1 μl of diluted cDNA (1:4) are added to a sterile microfuge tube or equivalent. The PCR is performed under the following thermocycling conditions: 2 min at 95 °C, 39 cycles of 15 s at 95 °C, 60 s at 60 °C, and cooled quickly to room temperature. Melting curve analysis is done by incubation at 60 °C to 95 °C at 0.1 °C s−1 with a smooth curve setting averaging 1 point. The melting temperatures for each product

To correct denomination ARGENTINA, BRAZIL, CHILE, COSAVE PARAGUAY, URUGUAY

69





Substantive

Substantive

are:Universal PPV detection (74 bp fragment): 80.08–81.52 °C.D strains (114 bp fragment): 84.3–84.43 °C.M strains (380 bp fragment): 85.34–86.11 °C.Internal control (181 bp fragment): 82.45–82.63 °C.4, 5, 6, 8 The use of the brand Invitrogen for first strand buffer, RNaseOUT TM , Superscript™ II and Platinum® Taq DNA high fidelity polymerase in this diagnostic protocol implies no approval of them to the exclusion of others that may also be suitable. This information is given for the convenience of users of this protocol and does not constitute an endorsement by the CPM of the chemical, reagent and/or equipment named. Equivalent products may be used if they can be shown to lead to the same results.7 The use of the brand Sigma for SYBR Green I in this diagnostic protocol implies no approval of them to the exclusion of others that may also be suitable. This information is given for the convenience of users of this protocol and does not constitute an endorsement by the CPM of the chemical, reagent and/or equipment named. Equivalent products may be used if they can be shown to lead to the same results.

Footnote added in relation to the use of brand names. See Argentina’s general comments

Footnote added in relation to the use of brand names. See Argentina’s general comments



226 3.3.4Real-time RT-PCR Mitigation

[55] A two-step RT-PCR protocol is used. The RT reaction is composed as

Ver comentario general en relación a las citas de las marcas

COSTA RICA

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Measures follows: 2 μl of 10 μM P1 primer, 2 μl of 10 μM Nad5-R primer, 4 μg total RNA and 5 μl water. Incubate 72 °C for 5 min, place on ice. Add 4 μl 5 × first strand buffer (Invitrogen), 2 μl 0.1 M DTT, 1 μl 10 mM dNTPs, 0.5 μl RNaseOUTTM (40 units μl−1) (Invitrogen), 1 μl Superscript™ II (Invitrogen) and 2.5 μl water. Incubate 42 °C for 60 min followed by 99 °C for 5 min. The 25 μl PCR reaction mixture is composed as follows: 400 nM PPV-U primer, 350 nM PPV-FM primer, 150 nM PPV-FD primer, 200 nM PPV-RR primer, 100 nM Nad5-F primer, 100 nM Nad5-R primer, 200 μM dNTPs, 2mM MgCl2, 1 × Karsai buffer (Karsai et al., 2002), 1:42 000 SYBR Green I (Sigma) and 0.1 μl Platinum® Taq DNA high fidelity polymerase (Invitrogen). The reaction mixture and 1 μl of diluted cDNA (1:4) are added to a sterile microfuge tube or equivalent. The PCR is performed under the following thermocycling conditions: 2 min at 95 °C, 39 cycles of 15 s at 95 °C, 60 s at 60 °C, and cooled quickly to room temperature. Melting curve analysis is done by incubation at 60 °C to 95 °C at 0.1 °C s−1 with a smooth curve setting averaging 1 point. The melting temperatures for each product are:Universal PPV detection (74 bp fragment): 80.08–81.52 °CD strains (114 bp fragment): 84.3–84.43 °CM strains (380 bp fragment): 85.34–86.11 °C

comerciales.


CUBA, PANAMA, PERU

71





Internal control (181 bp fragment): 82.45–82.63 °C.


[55] Sentence 4

Editorial ….2mM MgCl2, 1 × Karsai buffer (Karsai et al., 2002), …

subscript MgCl2 AUSTRALIA, SOUTH AFRICA


[55] Line 4

Line 7

Editorial RNaseOUTTMTM

MgCl22

RNaseOUTTM

MgCl22

EU, EPPO


[55] Last 4 lines

Substantive The temperature number as melting temperature should be an exact number instead of temperature range.

The melting temperature should be consistent with that in paragraph 84.

CHINA


[55] Sentence 6

Editorial and substantive

The PCR reaction mixture and 1 μl of diluted cDNA (1:4) are added to a sterile PCR microfuge tube or equivalent.

Sentence clarity. There is a difference between microfuge tubes and PCR tubes. PCR tubes are used for RT and PCR by design. Be specific.

JAMAICA


[55] Sentence 7

Editorial The PCR is performed under the following thermocycling conditions: 2 min at 95 °C, 39 cycles of 15 s at 95 °C, and 60 s at 60 °C, and then cooled quickly to room temperature.

‘and’ indicates the last input in the cycle. However, if the “cooled quickly to room temperature is an input in the cycle, please disregard this correction, but it must be stated clearly.

JAMAICA


[55] 8th Sentence

Substantive …, 39 cycles of 15 s at 95 °C, 60 s at 60 °C, and cooled quickly to room temperature 4 ºC.


JAPAN


[56] Editorial The method of Varga and James (2005) was evaluated using isolates of PPV-C, PPV-D, PPV-EA and PPV-M, and an uncharacterized strain, in Nicotiana and Prunus species.



This information is not relevant


COSTA RICA

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for the purposes of the Protocol. PANAMA, CUBA, PERU


[56] Line 2 Editorial ... strain, in Nicotiana and Prunus species.

Scientific name of species have to be in italic.

For appropriate formatting of genus names

EU, EPPO


235 4.Identification of Strains

[57] Technical 4. Identification of Strains The strain identification seems to lack some specificity and may not be reliable between laboratories

ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME


[57] Editorial Identification of sStrains No capitalization needed EU, EPPO


[58] Editorial The methods described in sections 3.2 and 3.3 for serological and molecular detection can also be used for identification of the virus. This section describes additional methods (DASI-ELISA, RT-PCR, Co-RT-PCR and real-time RT-PCR) for identification of PPV strains (see Figure 2).

This sentence is redundant ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME, TRINIDAD AND TOBAGO, JAMAICA


[58] Sentence 3

Editorial It is not necessary to determine which strain is present in order to identify PPV Strain identification is not a necessary component of PPV detection, but an NPPO may wish to determine the identity of the strain, for example, to assist in predicting its epidemiological behaviour of the virus.

Removal of text and addition of new text to improve clarity and meaning of the overall sentence.

CANADA


[58] Line 2 Technical ... methods (DASI or DAS-ELISA, RT-...

There are commercially available DAS-ELISA kits for D and M

EU, EPPO

73





characterisation (AMR Lab, Barcelona, Spain) based on the same antibodies than DASI-ELISA.


[58] Sentence 3

Editorial It is not necessary to determine which strain is present in order to identify PPV, but an NPPO may wish to determine the identity of the strain, for example, to assist in predicting its epidemiological behavior behaviour

More appropriate word MEXICO


[59]

Substantive

Given the variability of PPV, techniques other than sequencing or some PCR-based assays (see below) may provide erroneous results with a small percentage of isolates. However, it is generally possible to discriminate the D and M types of PPV using the serological or molecular techniques described below (Candresse and Cambra, 2006; Cambra et al., 2006a; Capote et al., 2006). In cases where the disease is not known to occur or for imported material, confirmations should be made with another more sensitive technique.

To confirm diagnosis, second test must be more sensitive than the first one.

Para confirmar un diagnostico, el Segundo test debe ser más sensible que el primero.

To confirm a diagnosis, the second test should be more sensitive than the first.


COSTA RICA

PANAMA, CUBA, PERU

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[59] Into the Figure, alter Serological tests, line 2

Technical DASI or DAS-ELISA with specific.. For agreement with paragraph 58 EU, EPPO

243 Figure 2 [60] Editorial Figure 6 2: Methods for the identification of strains of Plum pox virus.

To continue the figure number ARGENTINA, BRAZIL, CHILE, COSAVE PARAGUAY, URUGUAY

244 Figure 2 [60] Figure 2 Substantive New Box at the top of Figure 2 with following new text:Confirmed PPV Positive – by Universal ELISA and/or Universal RT-PCR

Add a new box at the top of Figure 2 with new text for better clarity. For completion and to avoid misunderstanding/confusion, the chart should indicate that the sample is a confirmed positive by ELISA using the universal PPV MAb 5b, or by the universal PPV RT-PCR with the P1 and P2 primers of Wetzel et al., 1991, before engaging in the complex process of strain typing.

CANADA

245 Figure 2 [60] At the end of Fig.2 legend

Editorial … identification of strains of Plum pox virus.

Full stop missing EU, EPPO

246 Figure 2 [60] The figure (top box)

Editorial Serological testDASI-ELISA with specific C-, D-, EA- or M M-specific monoclonal antibodies or

Molecular testRT-PCR (P1/PD/PM or mD5/mM3 primers), IC-RT-PCR (P1/PD/PM

General text improvements EU, EPPO

75





primers), Co-PCR (P10/P20/P1/P2 primers) and hybridization with D or M specific probes, or rReal-time RT-PCR (specific for C, D, EA, M or W types).

247 Figure 2 [60] 1. Substantive

2. Editorial

To add ‘Sequencing’ above ‘Serological test’ in the top box:Sequencing

Serological testDASI-ELISA with specific C, D, EA or M, Rec or T monoclonal antibodies; or

Sequencing methods should be added if it could be utilized to claryfy a strain quickly.

There is a statement about identification of ‘Rec’ and ‘T’ type in section 4.1.

JAPAN


[61]

Substantive

Further tests may be done in instances where the NPPO requires additional confidence in the identification of PPV type. Sequencing of the complete or partial CP gene may also be done, especially where atypical or undescribed types are present. PPV detection by ELISA test should be confirmed by PCR. In case of detection by PCR, it should be confirmed by sequencing.

Text was added to clarify



[61] Line 2 Substantive … of PPV type. Sequencing of the complete PPV genome or complete or partial CP, P3-6K1 and CI genes may also has to be done, …

The complete sequence of the PPV genome is the best when new types are detected. Currently there are numerous enterprises that are sequencing at a reasonable price (including deep-sequencing). Complete or partial sequence of CP, P3-6K1 and CI regions are recommended allowing PPV subgroup discrimination (Glasa et al., 2004. J Gen Virol. 85:2671-2681).

EU, EPPO

250 4.1Serological identification of

[64] Substantive This method has been validated in the DIAGPRO ring-test showing an

Removed because it is not relevant information for the

ARGENTINA, BRAZIL,

76





strains accuracy of 84% for PPV-D detection and 89% for PPV-M detection (Cambra et al., 2006b; Olmos et al., 2007). The 4D monoclonal antibody ...

purpose of this Draft



CHILE, COSAVE PARAGUAY, URUGUAY

COSTA RICA

PANAMA, CUBA, PERU

251 4.1Serological identification of strains

[64] 2nd sentence

Editorial The 4D monoclonal antibody is PPV-D specific but does not react against with all PPV-D isolates.

Text improvement EU, EPPO


[64] 3rd sentence

Editorial In addition, the monoclonal antibody AL used for PPV-M detection monoclonal antibody reacts with isolates belonging to strains M, Rec and T since these groups share the same coat protein sequence.



[65] Substantive DASI-ELISA kits based on the 4D (PPV-D specific) and AL (PPV-M, Rec and T specific) monoclonal antibodies are available from Agritest (Valenzano, Italy), AMR Lab (Barcelona, Spain) and Real/Durviz (Valencia, Spain).

Commercial brands removed.See general comments





COSTA RICA

EU, EPPO

PANAMA, CUBA, PERU

254 4.1Serological [65] At the end Substantive and Real/Durviz (Valencia, Spain). Additional information related to EPPO

77





identification of strains

of the paragrph

DAS-ELISA kits based on 4D and M monoclonal antibodies are available from AMR Lab (Barcelona, Spain).

previous paragraphs 58 and 59.



Editorial Move this paragraph after 63 and reword as follows [DASI-ELISA kits based on the monoclonal antibodies 4D (PPV-D specific) and AL (PPV-M, Rec and T specific) monoclonal antibodies are available from Agritest (Valenzano, Italy), AMR Lab (Barcelona, Spain) and Real/Durviz (Valencia, Spain).

The monoclonal antobody has not been mentioned before so it is more logic

EPPO


[66]

Substantive

Serological identification of PPV isolates from EA and C groups is done by DASI-ELISA using the EA- and/or the C-specific monoclonal antibodies described by Myrta et al. (1998, 2000), or others commercially available. These tests have not been validated and the antibodies are not commercially available.

Currently there are other monoclonal antibodies available for these strains



[66] Serological identification of PPV isolates from EA and C groups is done by DASI-ELISA using the EA- and/or the C-specific monoclonal antibodies described by Myrta et al. (1998, 2000). These tests need to be have not been validated and the antibodies are not commercially available.

Para reforzar la necesidad de que los test sean validados para su aplicación.

To reinforce the need of test validation for its application

COSTA RICA

PANAMA, CUBA, PERU

258 4.2.1 RT-PCR [70] Line 3 Editorial The 25 μl reaction mixture is composed as follows: 1 μM of P1 primer, 1 μM of either PD or PM primer, 250 μM dNTPs, 1 unit AMV reverse transcriptase (10 units μl−1), 0.5 units Taq DNA polymerase (5 units μl−1),

Correct formulation format ANTIGUA AND BARBUDA, AUSTRALIA, BARBADOS, DOMINICA, EU, EPPO,

78





2.5 μl 10 × Taq polymerase buffer, 1.5 mM MgCl22, 0.3% Triton X-100 and 2% formamide. The RT-PCR...

SOUTH AFRICA, SVG, SURINAME, THAILAND, TRINIDAD AND TOBAGO

259 4.2.1 RT-PCR [70]

Editorial

The 25 μl reaction mixture is composed as follows: 1 μM of P1 primer, 1 μM of either PD or PM primer, 250 μM dNTPs, 1 unit AMV reverse transcriptase (10 units μl−1), 0.5 units Taq DNA polymerase (5 units μl−1), 2.5 μl 10 × Taq polymerase buffer, 1.5 mM MgCl2, 0.3% Triton X-100 and 2% formamide and add 300 pg to 2 μg of RNA or DNA (specific detection and quantification of Plum Pox Virus by real time fluorescent reverse transcription PCR (Schneider et al 2004)). The RT-PCR ...

Text was added to clarify ARGENTINA, BRAZIL, CHILE, COSAVE PARAGUAY, URUGUAY

260 4.2.1 RT-PCR [70] The 25 μl reaction mixture is composed as follows: 1 μM of P1 primer, 1 μM of either PD or PM primer, 250 μM dNTPs, 1 unit AMV reverse transcriptase (10 units μl−1), 0.5 units Taq DNA polymerase (5 units μl−1), 2.5 μl 10 × Taq polymerase buffer, 1.5 mM MgCl2, 0.3% Triton X-100 and 2% formamide. The RT-PCR is performed under the following thermocycling conditions: 45 min at 42 °C, 2 min at 94 °C, 40 cycles of 30 s at 94 °C, 30 s at 60 °C, 1 min at 72 °C, followed by a final extension for 10 min at 72 °C and quickly cooled to room temperature. The PCR products are analysed by gel electrophoresis. The

Ver comentarios generales. COSTA RICA

79





P1/PD and P1/PM primers produce a 198 bp amplicon. The method was evaluated using six isolates of PPV-D and four PPV-M isolates.

261 4.2.1 RT-PCR [70] Sentence 1

Sentence 2

Substantive

Substantive

The 25 μl reaction mixture is composed as follows: 1 μM of P1 primer, 1 μM of either PD or PM primer, 250 μM dNTPs, 1 unit AMV reverse transcriptase (10 units μl−1), 0.5 units Taq DNA polymerase (5 units μl−1), 2.5 μl 10 × Taq polymerase buffer, 1.5 mM MgCl2, 0.3% Triton X-100 and 2% formamide.

The RT-PCR is performed under the following thermocycling conditions: 45 min at 42 °C, 2 min at 94 °C, 40 cycles of 30 s at 94 °C, 30 s at 60 °C, 1 min at 72 °C, followed by a final extension for 10 min at 72 °C and quickly cooled to room temperature.

- Specify the reaction mix to generate the first Chaín with oligos dT and the reaction mix to generate the second chain with reverse transcriptase. -Specify the concentration of RNA.

The thermocycling conditions to generation of first chain (with oligos dT) and generation of second chain with reverse transcriptase are mixed. - They must be separated.

MEXICO

262 4.2.1 RT-PCR [70] Last sentence

Editorial The method was evaluated using six isolates of PPV-D and four of PPV-M isolates.


263 4.2.1 RT-PCR [70] Sentence 2

Editorial ….thermocycling conditions: 45 min at 42 °C, 2 min at 94 °C, 40 cycles of 30 s at 94 °C, 30 s at 60 °C, and 1 min at 72 °C, followed by a final….

‘and’ indicates the last input in the cycle.

JAMAICA

264 4.2.1 RT-PCR [70] 2nd Sentence

Substantive …, 1 min at 72 °C, followed by a final extension for 10 min at 72 °C and quickly cooled to room temperature 4 ºC.


JAPAN

265 4.2.1 RT-PCR [70] The 25 μl reaction mixture is composed as follows: 1 μM of P1 primer, 1 μM of either PD or PM primer, 250 μM dNTPs, 1 unit AMV reverse

Same as paragraph 35: To complete the reaction. (it is necessary to add in the phrase “ and 5 µl of RNA”

PERÚ

80





transcriptase (10 units μl−1), 0.5 units Taq DNA polymerase (5 units μl−1), 2.5 μl 10 × Taq polymerase buffer, 1.5 mM MgCl2, 0.3% Triton X-100 and 2% formamide and 5 µl of RNA. The RT-PCR is performed under the following thermocycling conditions: 45 min at 42 °C, 2 min at 94 °C, 40 cycles of 30 s at 94 °C, 30 s at 60 °C, 1 min at 72 °C, followed by a final extension for 10 min at 72 °C and quickly cooled to room temperature. The PCR products are analysed by gel electrophoresis. The P1/PD and P1/PM primers produce a 198 bp amplicon. The method was evaluated using six isolates of PPV-D and four PPV-M isolates.

which represents the sample to be tested )

266 4.2.1 RT-PCR [71] Editorial PPV-Rec is identified using the mD5/mM3 Rec-specific primers described by Šubr et al et al. (2004): mD5 .....

Proper format ANTIGUA AND BARBUDA, BARBADOS, DOMINICA, SVG, SURINAME, TRINIDAD AND TOBAGO, JAMAICA

267 4.2.1 RT-PCR [72] Line 3 Editorial The 25 μl reaction mixture is composed as follows (modified from Šubr et al., 2004): 1 μM of each primer, 250 μM dNTPs, 1 unit AMV reverse transcriptase (10 units μl−1), 0.5 units Taq DNA polymerase (5 units μl−1), 2.5 μl 10 × Taq polymerase buffer, 2.5 mM MgCl22, 0.3% Triton X-100 and ....

Correct formulation format ANTIGUA AND BARBUDA, AUSTRALIA, BARBADOS, DOMINICA,EU, EPPO, SVG, SURINAME, THAILAND, TRINIDAD AND TOBAGO

81





268 4.2.2Immunocapture RT-PCR

[74] Sentence 2

Substantive The PCR reaction mixture is added directly to the coated microfuge PCR tubes.

There is a difference between microfuge tubes and PCR tubes.

JAMAICA

269 4.2.4 Real-time RT-PCR

[80] Line 1 1. Editorial2. Editorial

The primers and TaqMan probes used in the method of Capote et al et al. (2006) are:PPV-MGB-F primer .....

1. Correct format2. Correct punctuation

ANTIGUA AND BARBUDA, BARBADOS, DOMINICA,EU, EPPO, SVG, SURINAME, TRINIDAD AND TOBAGO, JAMAICA


[80] After paragraph 80

Substantive Add new paragraph after 80:It could be used primers and probe of an internal control for real-time RT-PCR protocol.

Same as paragraph 50a.

The internal control may be included to ensure the correct performance of the assay.

The TAQMAN reactions should included the internal control to give a level of confidence to the diagnostic, same as the real time reactions using SYBR green as pointed in the text of this protocol.

PERU


[81] The 25 μl reaction mixture is composed as follows: 1 μM of each primer, 150 nM MGB-D or MGB-M FAM probe, 1 × TaqMan Universal PCR Master Mix (Applied Biosystems9), and 1 × MultiScribe and RNase Inhibitor Mix (Applied Biosystems10). The reaction mixture and 5 μl of RNA template (see section 3.3), or spotted plant extracts (5 μl) or printed tissue sections (see section 3.3), are added to a

82





Substantive

Substantive

sterile microfuge tube or equivalent. The RT-PCR is performed under the following thermocycling conditions: 30 min at 48 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 60 s at 60 °C, and quickly cooled to room temperature. The PCR products are analysed in real time according to the manufacturer’s instructions. The method has been evaluated using 12 isolates each of PPV-D and PPV-M, and 14 samples co-infected with both types.9, 10 The use of the brand Applied Biosystems for FAM probe, TaqMan Universal PCR Master Mix, and for MultiScribe and RNase Inhibitor Mix in this diagnostic protocol implies no approval of them to the exclusion of others that may also be suitable. This information is given for the convenience of users of this protocol and does not constitute an endorsement by the CPM of the chemical, reagent and/or equipment named. Equivalent products may be used if they can be shown to lead to the same results.


Este párrafo no aporta información relevante para el protocolo.


ARGENTINA, BRAZIL, CHILE, COSAVE PARAGUAY, URUGUAYCOSTA RICA



[81] The 25 μl reaction mixture is composed as follows: 1 μM of each primer, 150 nM MGB-D or MGB-M FAM probe, 1 × TaqMan Universal PCR Master Mix (Applied Biosystems), and 1 × MultiScribe and RNase Inhibitor Mix (Applied Biosystems). The reaction mixture and 5 μl of RNA template (see section 3.3), or spotted plant extracts (5 μl) or printed tissue sections (see

See general comments regarding the use of brand names.This information is not relevant for the purposes of the Protocol.

CUBA, PANAMA, PERU

83





section 3.3), are added to a sterile microfuge tube or equivalent. The RT-PCR is performed under the following thermocycling conditions: 30 min at 48 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 60 s at 60 °C, and quickly cooled to room temperature. The PCR products are analysed in real time according to the manufacturer’s instructions. The method has been evaluated using 12 isolates each of PPV-D and PPV-M, and 14 samples co-infected with both types.



Substantive - The final volumes and reagent volumes are not clearly described. It is not clear if the final reaction volume is 25 ul or 30 ul. The authors should indicate the specific reagent volumes for ease of replication and implementation.

CANADA


[81] Sentence 3

Editorial ….following thermocycling conditions: 30 min at 48 °C, 10 min at 95 °C, 40 cycles of 15 s at 95 °C, and 60 s at 60 °C, and quickly….

‘and’ indicates the last input in the cycle.

JAMAICA


[81] 3rd Sentence

Substantive ..., 10 min at 95 °C, 40 cycles of 15 s at 95 °C, 60 s at 60 °C, and quickly cooled to room temperature 4 ºC.


JAPAN


[82] Line 2 at the end

Editorial PPV-C, PPV-EA and PPV-W are specifically identified using SYBR Green I chemistry according to the method of Varga and James (2006). The primers used in this method are:P1 .......

Correct punctuation ANTIGUA AND BARBUDA, BARBADOS, DOMINICA,EU, EPPO, SVG, SURINAME, TRINIDAD

84





AND TOBAGO


[84] Line 4

Line 10 at the end

1. Editorial

2. Editorial

3. Editorial

The 25 μl RT-PCR reaction is composed as follows: 2.5 μl of a 1/10 water dilution of extracted RNA (see section 3.3) and 22.5 μl of master mix. The master mix has the following composition: 2.5 μl of Karsai Buffer (Karsai et al., 2002); 0.5 μl each of 5 μM primers PPV-U, PPV-RR or P1, Nad5R and Nad5F; 0.5 μl of 10 mM dNTPs; 1 μl of 50 mM MgCl22; 0.2 μl of RNaseOUT™ (40 units μl−1; Invitrogen); .... The melting temperatures for each product are:C strain (74 bp fragment): 79.84 °CEA strain (74 bp fragment): 81.27 °CW strain (74 bp fragment): 80.68 °C.

1. correct formulation format

2. correct punctuation

3. correct punctuation

ANTIGUA AND BARBUDA, BARBADOS, DOMINICA,SVG, SURINAME


[84] Sentence 2

Editorial The master mix has the following composition: 2.5 μl of Karsai Buffer (Karsai et al., 2002); 0.5 μl each of 5 μM primers PPV-U, PPV-RR or P1, Nad5R and Nad5F; 0.5 μl of 10 mM dNTPs; 1 μl of 50 mM MgCl22; 0.2 μl of RNaseOUT™ (40 units μl−1; Invitrogen); 0.1 μl of Superscript™ III (200 units μl-1; Invitrogen); 0.1 μl of Platinum® Taq DNA high fidelity polymerase (5 units μl-1, Invitrogen); and 1 μl of 1:5 000 (in TE, pH 7.5) SYBR Green I (Sigma) in 16.1 μl water.

Correct formulation format. AUSTRALIA, EU, SOUTH AFRICA, TRINIDAD AND TOBAGO


[84] Line 4

Line 10 at the end

Editorial

MgCl22

averaging 1 point. The melting temperatures for each product are:

MgCl2

:

EPPO

280 4.2.4 Real-time RT- [84] Sentence Editorial The melting temperatures for each Correct punctuation. TRINIDAD

85





PCR 5 product are:C strain (74 bp fragment): 79.84 °CEA strain (74 bp fragment): 81.27 °CW strain (74 bp fragment): 80.68 °C.

Close bracket. AND TOBAGO


[84] The 25 μl RT-PCR reaction is composed as follows: 2.5 μl of a 1/10 water dilution of extracted RNA (see section 3.3) and 22.5 μl of master mix. The master mix has the following composition: 2.5 μl of Karsai Buffer (Karsai et al., 2002); 0.5 μl each of 5 μM primers PPV-U, PPV-RR or P1, Nad5R and Nad5F; 0.5 μl of 10 mM dNTPs; 1 μl of 50 mM MgCl2; 0.2 μl of RNaseOUT™ (40 units μl−1; Invitrogen); 0.1 μl of Superscript™ III (200 units μl-1; Invitrogen); 0.1 μl of Platinum® Taq DNA high fidelity polymerase (5 units μl-1, Invitrogen); and 1 μl of 1:5 000 (in TE, pH 7.5) SYBR Green I (Sigma) in 16.1 μl water. The reaction is performed under the following thermocycling conditions: 10 min at 50 °C, 2 min at 95 °C, 29 cycles of 15 s at 95 °C, and 60 s at 60 °C. Melting curve analysis is performed by incubation at 60 °C to 95 °C at 0.1 °C s−1 melt rates with a smooth curve setting averaging 1 point. The melting temperatures for each product areC strain (74 bp fragment): 79.84 °CEA strain (74 bp fragment: 81.27 °CW strain (74 bp fragment): 80.68 °C.



COSTA RICA

CUBA, PANAMA, PERU


[84] The 25 μl RT-PCR reaction is composed as follows: 2.5 μl of a 1/10 water dilution of extracted RNA (see

ARGENTINA, BRAZIL, CHILE,

86





Substantive

Editorial

Substantive

section 3.3) and 22.5 μl of master mix. The master mix has the following composition: 2.5 μl of Karsai Buffer (Karsai et al., 2002); 0.5 μl each of 5 μM primers PPV-U, PPV-RR or P1, Nad5R and Nad5F; 0.5 μl of 10 mM dNTPs; 1 μl of 50 mM MgCl2; 0.2 μl of RNaseOUT™ (40 units μl−1; Invitrogen11); 0.1 μl of Superscript™ III (200 units μl-1; Invitrogen12); 0.1 μl of Platinum® Taq DNA high fidelity polymerase (5 units μl-1, Invitrogen13); and 1 μl of 1:5 000 (in TE, pH 7.5) SYBR Green I (Sigma14) in 16.1 μl water. The reaction is performed under the following thermocycling conditions: 10 min at 50 °C, 2 min at 95 °C, 29 cycles of 15 s at 95 °C, and 60 s at 60 °C. Melting curve analysis is performed by incubation at 60 °C to 95 °C at 0.1 °C s−1 melt rates with a smooth curve setting averaging 1 point. The melting temperatures for each product areC strain (74 bp fragment): 79.84 °CEA strain (74 bp fragment) : 81.27 °CW strain (74 bp fragment): 80.68 °C.11, 12, 13 The use of the brand Invitrogen for RNaseOUT TM , Superscript™ III and Platinum® Taq DNA high fidelity polymerase in this diagnostic protocol implies no approval of them to the exclusion of others that may also be suitable. This information is given for the convenience of users of this protocol and does not constitute an endorsement by the CPM of the chemical, reagent and/or equipment


Footnote added in relation to the use of brand names. See general

COSAVE PARAGUAY, URUGUAY

87





named. Equivalent products may be used if they can be shown to lead to the same results.14 The use of the brand Sigma for SYBR Green I in this diagnostic protocol implies no approval of them to the exclusion of others that may also be suitable. This information is given for the convenience of users of this protocol and does not constitute an endorsement by the CPM of the chemical, reagent and/or equipment named. Equivalent products may be used if they can be shown to lead to the same results.

comments


[85] Sentence 1

Editorial This method was evaluated using an one isolate from each of PPV-C, PPV-D, PPV-EA and PPV-W.

Re-worded JAMAICA

284 5. Records [87] Sentence 1

Editorial The records required to be kept are listed in section 2.5 of ISPM 27: 2006.

For consistency with new referencing style


285 5. Records [88] Line 7 Substantive …tissue sections on paper or nylon on membranes should be kept at room temperature.

Technical improvement and clarification

EU, EPPO

286 5. Records [88] 1st sentence

Substantive additional material should be kept for at least one year

To be used in arbitration if necessary

EU, EPPO

287 5. Records [88] 1st to 3rd indents

Substantive -The original sample (labelled appropriately for traceability) should be kept frozen at −80 °C −20 °C or freeze-dried and kept at room temperature.-If relevant, RNA extractions should be kept at −80 °C −20 °C and/or spotted plant extracts or printed tissue sections paper on membranes should be kept at room temperature.-If relevant, RT-PCR amplification

Those substances can be stored in frozen temperature (-20 oC).

JAPAN

88





products should be kept at −80 °C −20 °C .

288 5. Records [88] As new indent 4

Substantive - RNA preservatives To further assist laboratories without large freezers for storage and also allows for easy movement between laboratorios

SOUTH AFRICA

289 6.Contact Points for Further Information

[90] Sentence 1

Editorial Equipe de Virologie, Institut National de la Recherche Agronomique (INRA),

Add comma in first line of address

AUSTRALIA


[90] Editorial

Editorial

F-33883

d´’Ornon

Consistency with the other French code postal in paragraph [96]. NB: In some countries, it is recommended to make precede the postal code by the letter characterizing the foreign country, in this case F.

Error of character

EU, EPPO


[90] Add new contact

Substantive APHIS PPQ PHP RIPPSMolecular Diagnostic LaboratoryBARC Building 580Powder Mill Road, Beltsville, MD20705Phone: 301-504-5700Fax: 301-504-6124

The USDA PPQ laboratory in Beltsville, Maryland, has a lot of experience in this area and would be a good contact point for countries that would need guidance in implementing the molecular protocol for PPV.

USA


[93] All paragraph

Editorial Instituto Valenciano de Investigaciones Agrarias (IVIA), Plant Protection and Biotechnology Centre, Carretera Moncada-Náquera km 5, 46113 Moncada (Valencia), Spain (Dr. Mariano Cambra, e-mail: [email protected]; Tel.: +34 963424000; Fax: +34 963424001).

Corrected versión in agreement with the editorial form of the other contact points.

EU, EPPO


[95] Sentence 1

Editorial Sidney Laboratory, Canadian Food Inspection Agency (CFIA), British Columbia, V8L 1H3 Sidney, Canada

The e-mail address is incorrect and needs to be updated.

CANADA

89





(Dr. Delano James, e-mail: [email protected] [email protected]; Tel.: +1 250 3636650; Fax: +1 250 3636661).


[96] Editorial

Editorial

Interprofessionnel

BP 21

One “n” is missing

The blank is missing

EU, EPPO

295 7.Acknowledgements [98] Sentence 1

Editorial Dr Mr N.L. Africander, National Department of Agriculture Department of Agriculture, Forestry and Fisheries, Private Bag X 5015, Stellenbosch, 75999, South Africa.

Correct title, address and institution of the technical member from South Africa

SOUTH AFRICA

296 8. References [99] Substantive The disease was described as a viral disease in 1932 by Atanasoff.D. 1932 j.univ. of Sofia. Why this is not mentioned in the list of referentes.

JORDAN

297 8. References [106] Line 1 Substantive Capote, N., Bertolini, E., Olmos, A., Vidal, E., Martínez, M.C. & Cambra, M. 2009. Direct

The name of one of the authors (Vidal, E.,) was missing.

EU, EPPO

298 8. References [122]

Editorial

Osman, F. & Rowhani, A. 2006. Application of a spotting sample preparation technique for the detection of pathogens in woody plants by RT-PCR and real-time PCR (TaqMan). Journal of Virological Methods, 133: 130–136.

Rosner, A., Shilboleth, Y., Spiegel, S., Krisbai, L., Kölber, M ., 1998. Evaluating the use of immunocapture and sap-dilution PCR for the detectionof Prunus necrotic ringspot virus. Acta Horticulturae 472, 227-233

Reference is required


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